To demonstrate that the MISER-GC-MS method can be readily applied to other biological, enzyme as well as reaction systems, the screening was applied to a shuffled library (unpublished results) of fungal unspecific peroxygenases (UPO), which can be heterologously secreted from S. cerevisiae and catalyse amongst other reactions the hydroxylation and further oxidation of 1, 2,3,4-tetrahydro-naphthalene (tetralin). Whereas the previous case study proved to allow the simultaneous quantification of the internal standard and the product, we here aimed toward the analysis of three molecules with one sample injection every 33 s. To determine the feasibility of the quantification of three molecules by MISER-GC-MS the samples 1,2,3,4tetrahydronaphthol, α-tetralone and 1-naphthol were injected in three different concentrations and the MS response compared to the obtained values when injecting only one analyte at a time (Figure ). The data for each analyte revealed excellent correlation to the analysis with the simultaneous analysis of all molecules, hence allowing the desired simultaneous analysis of the three target molecules. One previously identified UPO variant was 96 times expressed (biological replicates) in microtiter plate format and the corresponding UPO containing supernatant tested for the conversion of tetralin and analysed by MISER-GC-MS. The aim was to validate that the MISER-GC-MS technology enabled the analysis of the entire plate with the individually expressed variant with an overall standard deviation of less than 10 %. We were delighted to see that the analysis of the entire 96 well plate within 48 minutes showed a standard deviation of only 9.7 % for the formation of 1,2,3,4-tetrahydro-naphthol (Figure ). We now created a chimera library consisting of 243 possible gene combinations and screened 672 transformants produced and secreted in S. cerevisiae supernatant. In the seven different 96 well plates, four previously discovered novel, active UPO variants have been placed. All four variants could be identified within the screening as well as >30 newly identified hits (Figure ). In order to be able to deconvolute the actual protein amount from the protein activity within a single microtiter plate, each variant was equipped with a C-terminal fused split-GFP tag (GFP-11) resulting in a quantitative fluorescence signal upon recombination with GFP-1-10. Correlation of GC-MS with split-GFP data revealed several hits showing a decreased secretion compared to PaDa-I as WT and decreased activity but yielding a comparable or even higher activity-to-protein-amount factor. Due to the resulting huge amount of data and to ensure high quality and correct peak integration in each microtiter plate, an automatised R-script has been written. The script assesses the data from the MISERgram for reproducibility (based on internal standard) and based on the injection interval whether every peak has been correctly integrated or injected. All data are displayed as chromatogram were each m/z signal can be individually selected and zoomed in via hover-visualization. The quotient of internal standard and product peak is built and illustrated as bar chart and colour-coded microtiter plate for fast data evaluation. Lastly, the data are correlated to the split-GFP signal giving an expression independent activity signal. All data are shown in one final output html file.