The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIPA). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1-aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (maximum inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metabolism. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (maximum inhibition of <50%) or no apparent inhibitory effects in the HTS assay. In the 96-well plate induction assay, the CYP3A4 inducers rifampin, phenobarbital, carbamazepine, phenytoin, troglitazone, rosiglitazone, and pioglitazone yielded dose-dependent induction of LIPA metabolism, whereas the CYP1A2 inducers omeprazole and 3-methylcholanthrene did not display any induction in the CYP3A4 activity. The high sensitivity and specificity of the assays, the relative ease of execution, and reduced cost, time, and test material requirements suggest that the HTS assays may be applied routinely for screening a large number of chemicals in the drug discovery phase for CYP3A4 inhibitory and inducing potential.