A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 3A4/5 in human liver microsomes is described. In contrast to the conventional testosterone 6beta-hydroxylation assay, the new method does not require high-performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP3A4/5-mediated 6beta-hydroxylation of testosterone labeled with tritium in the 6beta position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. Using commercially available [1,2,6,7-(3)H]testosterone as substrate, we demonstrated that the reaction is NADPH-dependent, and sensitive to CYP3A4/5/5 inhibitors and a CYP3A4/5/5-specific inhibitory monoclonal antibody, but not to inhibitors of or antibodies against other P450 enzymes. The method was further improved by synthesis of testosterone specifically tritiated in the 6beta position, which displayed greatly improved conversion rate with an ensuing increase in assay sensitivity. Competition experiments using tritiated and unlabeled testosterone indicated that CYP3A4/5-mediated 6beta-hydroxylation exhibits positive cooperativity and a modest kinetic isotope effect. IC(50) values for more than 40 structurally diverse chemical inhibitors were not significantly different from those determined in the testosterone 6beta-hydroxylation assay, using HPLC-tandem mass spectrometry analysis. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classical testosterone 6beta-hydroxylation assay, which is the most widely used method to assess the potential for CYP3A4/5 inhibition of new chemical entities.