Directed evolution mimics the natural selection process of Darwinian evolution in the laboratory. Random gene libraries are created and the corresponding enzyme variants are assessed for their altered and desired properties. The improved variants are thereafter submitted to further rounds of mutagenesis and screening. The library size in a classical directed evolution approach consist of 500 -2000 variants per round requiring a rapid analytical tool to quantify the activity of the different enzyme variants. Highly sensitive and robust assays are needed since starting activities are often below 10 turnovers (TON) and the reactions need to be screened in complex biological matrices i.e. cell lysates, whole cell systems or cultivation supernatants. The overwhelming majority of successfully employed enzyme assays relies on changes in absorption or fluorescence wavelengths during the course of the reaction of either participating model substrates or of additionally coupled enzyme cascade reactions. To further expand the portfolio of assaying techniques, a flexible, substrate unspecific analytical tool would be of high interest since UV/vis or fluorescence-based assays suffer from the requirement of spectroscopically active compounds. These fit-for-purpose compounds, however, can differ substantially from the actual substrate of interest yielding enhanced variants for a differing substrate conversion. The ideal assay would allow the screening with the exact substrate of interest, be highly sensitive as well as exceedingly reproducible. A flexible and sensitive analytical technique is provided by chromatographic technologies such as liquid (LC) or gas chromatography (GC). While these techniques often provide the necessary sensitivity and are applicable to a wide range of substrates, they suffer from long analysis times preventing a highthroughput screening with several hundreds of samples. Developments in the last 15 years delivered faster chromatographic technologies, like the UHPLC (ultra high performance liquid chromatography) reducing analytical time by using sub-2-micron particle in their packed columns and high pressure of up to 1000 bar. A system capable of high-throughput GC analysis was introduced by Boeker et al.: the flow field thermal gradient gas chromatography (FF-TG-GC). The first prototype of an FF-TG-GC instrument was constructed in 2015 by combining the classical temperature gradient with an additional spatial temperature gradient. This approach is based on theoretical calculations of heat transport processes and led to a fast analysis in less than 60 seconds for one sample finding its application for example at lowered temperatures for explosive substances. The analysis of multiple, overlapping samples in one chromatogram, coined multiplexing, has been introduced in 1967 by Izawa for GC and was extended by Trapp and co-workers for a high-throughput approach. This technique bases on a high number of short injections in fast succession, in which the pattern for all sample injections is coded by a pseudo-random binary sequence derived from Hadamard matrices. This pattern allows decoding of the resulting highly overlapping peaks in a so-called multiplexed chromatogram into single chromatograms and represents a very powerful technology. Trapp et al. were able to show a throughput enhancement factor of 38. The method relies on short but highly reproducible injections with regard to injection length, time and volume. They therefore employed a specifically [a] Anja Knorrscheidt, Pascal Püllmann, Eugen Schell, Dominik Homann, Jun.-Prof. Dr. Martin J. Weissenborn Leibniz Institute of Plant Biochemistry Bioorganic Chemistry Weinberg 3, 06120 Halle (Saale), Germany E-mail: martin.weissenborn@ipb-halle.de [b] Dr. Erik Freier Leibniz-Institut für Analytische Wissenschaften -ISAS-e.V. Otto-Hahn-Str. 6b, 44227 Dortmund, Germany [c] Jun.-Prof. Dr. Martin J. Weissenborn Institute of Chemistry, Martin-Luther-University Halle-Wittenberg Kurt-Mothes-Str. 2, 06120 Halle (Saale), Germany Supporting information for this article is given via a link at the end of the document built, highly-sophisticated injector system to achieve this throughput enhancement. Welch et al. introduced MISER-multiple injections in a single experimental run-in 2010 as a method to enhance throughput in LC measurements without the need of special equipment or expert knowledge. The MISER technique relies on injecting several different samples under isocratic conditions into one chromatography run requiring baseline separation of the consecutive peaks. This enabled the performance of long chromatographic separations in a high-throughput manner as usually peak-free ("silent") areas of the chromatogram are used for multiple injections. While MISER was originally published as LC technique, it has already been extended to LCMS and was applied in kinetic measurements, analysis of enantiopurity, and determining ingredients in different matrices. As a screening device for directed evolution, we required a medium to high throughput (>500 samples/day) technique, which is sensitive-down to low µM product concentrations-and allows the analysis of a broad range of products. The method of MISER-GC-MS has not yet been employed for these applications or large sample numbers, but its properties seem highly suitable for the application in directed evolution for several reasons: i) the quantification of target substances by GC-MS extracted from crude lysates, cell supernatants or whole cells is far less prone to ion suppression by matrix effects than by LC-MS systems, ii) MISER allows a distinct throughput enhancement for GC without the requirement of special equipment, iii) it can be combined with internal standards to compensate for deviations in injection time, volume and length, or even in concentration due to sample evaporation, iv) MISER-GC-MS also enables the quantification of multiple molecules with overlapping peak areas, eliminating the need for chromatographical separation and therefore raising the throughput enhancement factor further and v) the MS-based detection is highly sensitive and allows low range detections. Herein, we report the development of a new MISER-GC-MS strategy paving the way for an assay-independent platform for enzymatic reactions applicable for any GC-MS equipped with an autosampler (Figure ). We have performed MISER-GC-MS in the screening of a non-natural carbene-transfer reaction in Escherichia coli (E. coli) leading to the discovery of tryptophan as an axial heme ligand. To demonstrate the versatility of the MISER-GC-MS system a second reaction in a different biological system has been investigated: The fungal unspecific peroxygenase (UPO) catalysing a hydroxylation reaction performed in yeast cell (S. cerevisiae) supernatant. In the latter example 672 transformants (including controls) have been screened within a day using a sample injection frequency of 33 s while quantifying three molecules in parallel and thereafter normalising the enzyme concentration with a split-GFP assay. To further facilitate the data evaluation, a freely available R-script has been written allowing the analysis, quality control and correlation to split-GFP signal in "one click".