A rapid quantitative assay method, developed by combining fast gradient liquid chromatography and electrospray ionization-ion trap mass spectrometry, is described for the simultaneous determination of CYP450 probe substrate metabolites (4-aminophenol for CYP2E1, acetaminophen for CYP1A2, dextrorphan for CYP2D6, 4'-hydroxymephenytoin for CYP2C19, 4-hydroxytolbutamide for CYP2C9 and 6β-hydroxytestosterone for CYP3A4) in microsomal incubations. Using this method Michaelis-Menten kinetic parameters K m and V m a x for the probe substrates in human liver microsomes were obtained. This LC-MS-MS method, developed with the use of LC-ESI-ion trap MS instrumentation, can efficiently be used to improve the throughput and cost-effectiveness in the preclinical drug metabolism studies.