In this study, a simple in vitro method for detecting human P450 (CYP) quasi-irreversible and irreversible inhibitors was evaluated. For the method, cDNA-expressed CYPs were applied to microtiter plate assays, CYP inhibitors were co-incubated with fluorometric substrates, and IC(50) were continuously measured (without stopping enzyme reactions). The typical reversible inhibitors (sulfaphenazole, tranylcypromine, quinidine, ketoconazole) showed constant IC(50) throughout the reaction. In contrast, the typical quasi-irrversible inhibitors (isosafrole, erythromycin, troleandomycin, diltiazem) and the typical irreversible inhibitors (furafylline, propranolol, mifepristone) showed time-dependent decreases in IC(50). For CYP3A4 inhibition studies, two substrates, 7-benzyloxyresorufin (BzRes) and 7-benzyloxy-4-trifluoromethyl-coumarin (BFC), were used. The IC(50) of the CYP3A4 inhibitors were dependent on the substrate. However, the quasi-irreversible and irreversible inhibitors could be detected by examining changes in the IC(50), regardless of the substrate. Further, the detection method was applied to josamycin and bergamottin. Josamycin did not show definite time-dependent decreases in IC(50) for CYP 3A4, suggesting that josamycin is neither a quasi-irrversible nor an irreversible inhibitor of CYP3A4. On the other hand, bergamottin showed time-dependent decreases in IC(50) for CYP1A2, CYP 2C9, CYP 2C19, CYP 2D6 and CYP 3A4, suggesting that bergamottin is a quasi-irrversible or an irreversible inhibitor of the 5 CYP isoforms. This method provides more rapid and reliable detection of quasi-irreversible and irreversible inhibitors and may be useful in drug discovery.