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Direct injection of samples on high-resolving mass spectrometers is an effective way to maximize analytical throughput and yet allow analyte discrimination in complex samples by mass-to-charge ratio. We present a platform of flow injection electrospray–time-of-flight mass spectrometry to profile small molecules in >1400 biological extracts per day at native mass resolution. We comprehensively benchmark the performance with more than 5000 injections of chemically defined standards and Escherichia coli cellular extracts obtained from miniscale cultivations. For at least 90% of tested compounds, we attain a linear response over 3 decades of concentration, interday coefficient of variation of <20%, and a mass accuracy of <0.001 amu. In polar Escherichia coli fractions, we reproducibly detected >1500 distinct ions in each mode. The accurate mass and correlation analysis enabled one to assign with good …