A refined cytochrome P450 (P450) enzyme IC₅₀ shift assay for more accurately screening CYP3A time-dependent inhibitors (TDIs) is presented. In contrast to the regular IC₅₀ shift assay, in which only one pair of P450 inhibition curves is generated, this modified method generates two pairs of inhibition curves; one pair of curves is created from human liver microsomal incubations with the test article in the presence or absence of NADPH (curves 1 and 2) (same as the traditional assay), and the other pair is created from new microsomal incubations with extract (compound/metabolites) of previous incubations (curves 3 and 4). To assess the true CYP3A time-dependent inhibition, we propose a new parameter, the vertical IC₅₀ curve shift (VICS), represented by vertical shift difference between the two sets of curves divided by inhibitor concentration at which maximal vertical shift of curves 1 and 2 is observed. A shift in the curves 1 and 2 could mean a time-dependent inhibition or formation of a more active inhibitory metabolite(s). The new method provides more reliable characterization of the shift as a result of a true TDI- or metabolite-mediated reversible inhibition. Nine known TDI drugs were evaluated using this refined shift assay. The derived VICS values correlated well with the reported k(inact)/K(I) values derived via the conventional dilution assay method. Thus, the refined assay can be used to identify a true TDI and quantitatively assess the inactivation potential of TDIs in a high-throughput fashion. This assay can be invaluable to screen for true P450 TDIs in the early drug discovery.