In this study we have evaluated the reliability of a fluorescence-based method used for rapid identification of irreversible CYP inhibitors (mechanism-based inhibitors). This was accomplished by comparing the time-dependence pattern of IC50 values from fluorometric kinetic measurements. For irreversible CYP inhibitors, IC50 values decreased as incubation proceeded. This was due to progressive inactivation of corresponding enzymes by reactive metabolites generated during the incubation. This change pattern was confirmed using a number of known irreversible CYP inhibitors, including furafylline, midazolam, erythromycin, clarithromycin, oleandomycin, 17alpha-ethynylestradiol and verapamil. The pattern was different in reversible inhibition, depending upon the compounds tested in the fluorometric kinetic assay. For some compounds, such as clotrimazole, IC50 values remained relatively stable, whereas other compounds, such as miconazole, terfenadine and ketoconazole showed a significant increase with incubation time. Monitoring tested compounds by LC-MS/MS during the incubation confirmed that increases of IC50 were probably caused by the loss of inhibitors, resulting from either metabolic degradation, or non-specific binding to microsomal proteins.