A generic method employing ultrafast liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for routine screening of drug candidates for inhibition of five major human cytochrome P450 (CYP) isozymes, CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. The method utilized a monolithic silica rod column to allow fast flow rates to significantly reduce chromatographic run time. The major metabolites of six CYP-specific probe substrates for the five P450 isoforms were monitored and quantified to determine IC50 values of five drug compounds against each P450 isozyme. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. Each pooled sample containing six substrates and an internal standard was separated and detected in only 24 seconds. The combination of ultrafast chromatography and sample pooling techniques has significantly increased sample throughput and shortened assay turnaround time, allowing a large number of compounds to be screened rapidly for potential P450 inhibitory activity, to aid in compound selection and optimization in drug discovery. Copyright © 2003 John Wiley & Sons, Ltd.