A standard autosampler (AOC-5000, Shimadzu, Kyoto, JP) was modified for fast injection application by decoupling the autosampler from the GC instrument. The read-out-signal of the GC was suppressed enabling independently controlling of the autosampler. The method development commenced by altering various conditions at the autosampler. The various settings like post-cleaning with isopropanol after each injection, filling speed (3 µL/s) and filling strokes for each microtiter plate led to significantly improved standard deviations from initially 51.6 %no post-cleaning, 10 µL/s filling speed, no filling strokes-to 6.5 % (Table ). The data were achieved using GC conditions of 230 °C and a split ratio of 60. The GC-MS system was then further developed for the best MISER conditions using the product of an enzymatic reaction of interest, ethyl 3-indoleacetate, as well as methyl indole-3-carboxylate as internal standard. MISER-GC-MS method development was performed employing three different ethyl 3-indoleacetate concentrations (20, 50 and 90 µM) in triplicates. Due to the continuous injection of up to 96 samples into one experimental run, the methods have to be performed under an isocratic temperature profile. Further parameters, which were investigated were the split ratio, the MS mode (SIM or Scan) and the injection interval (Figure , Table , Figure ). The lowest oven temperature of 150 °C led to extensive peak tailing and poor baseline separation therefore leading to problems in automated area integration. Utilising an temperature of 190 °C excellent baseline separation and enhanced peak shape could be obtained as well as an improved standard deviation at 20 µM ethyl 3-indoleacetate from 8.6 % to 4.0 %. In order to position the injection peak, between different analytes peaks, the injection interval was altered from 67 s to 82 s. This further lowered the standard deviation and allowed the increase of the split ratio up to 60 resulting in excellent standard deviations of 1.0 % for 20 µM ethyl 3-indoleacetate. With these optimised analytical conditions in hand, we further challenged the system by using E. coli cell lysates spiked with ethyl 3-indoleacetate and analysed the resulting samples in 96well experiments using methyl indole-3-carboxylate as the internal standard. As further quality control and for calibration purposes ethyl 3-indoleacetate (20 µM) extracted from a buffer system was injected after each microtiter plate row (12 samples) and after 96 wells calibration standards (2 x 20 µM, 2 x 50 µM, 2 x 90 µM) were injected as well. The system was assessed with the best conditions of the GC system (190 °C, split ratio 60, and 82 s injection) leading to a standard deviation of 4.0 % (Figure , left). This could be even further improved to a standard deviation of 2.5 % by increasing the temperature to 230 °C that moreover allowed a shorter injection interval of 33 s (Figure , middle, Table ). Since the developed methods shall be applied in the screening of non-natural enzyme activities, which in general suffer from low turnovers and require high substrate loading, the contamination of the GC-column could pose a substantial problem within 96 injections. Therefore an additional method, which includes a heating cycle after 12 injections (one microtiter plate row) has been developed as previously shown by