The application of ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) for high-throughput analysis of a 96-well plate based metabolic stability assay has been investigated. Full-scan data were acquired, with run times of 2.5–3.5 min, from which narrow window extracted ion chromatograms were generated, producing quantitative data for the test compound equivalent to that obtained by high-performance liquid chromatography with tandem mass spectrometric detection on a triple quadrupole instrument (HPLC/MS/MS). Sensitivity and mass accuracy were maintained over the analysis of >300 samples. Additionally, the UPLC/TOFMS datasets obtained gave access to metabolic route information, at no cost in terms of sensitivity for the test compound. Copyright © 2006 John Wiley & Sons, Ltd.