BACKGROUND The traditional in vitro approach for assessing potential CYP induction has been to simply compare changes in CYP activities using known CYP-specific probe substrates following exposure to the test compound to that of vehicle and/or positive controls in primary cultured human hepatocytes. The objective of these current studies was to develop and implement a highly efficient 96-well CYP induction assay in which mRNA levels, protein levels, and the conventional enzyme activities of CYP1A2, CYP2B6, and CYP3A4/5 are all measured in the same well after 48 h. Cytotoxicity is also assessed in the same well after 24 and 48 h of incubation. Since enzymatic activity data alone often 'misses' CYP induction due to compounding factors, such as CYP mechanism-based inactivation, this 'all-inclusive' approach efficiently maximizes the generation of additional useful and comprehensive data. This data can more readily identify potential CYP induction liabilities in the drug discovery process and, therefore, avoid potential drug-drug interactions in the clinic. METHODS One 96-well plate with cryopreserved human hepatocytes accommodated up to nine test compounds at three clinically relevant concentrations, positive and negative controls for CYP1A2, CYP2B6, and CYP3A4/5, and a vehicle control (0.1% DMSO) in three different lots of cryopreserved human hepatocytes. Ritonavir, a positive control for CYP3A inactivation/induction, and staurosporine, a positive control for cytotoxicity, were included. The compounds 3-methylcholanthrene (a CYP1A2 inducer), phenobarbital (a CYP2B6 inducer), and rifampicin (a CYP3A4/5 inducer) served as positive controls. RESULTS Data showed a strong correlation between the fold-increases in CYP activity, mRNA level, and protein level after incubation of the CYP isoforms with positive controls compared to the vehicle control. Ritonavir resulted in a decrease in CYP3A/5 activity, yet a concomitant increase in mRNA and protein levels of CYP3A4. Cytotoxicity was positive for staurosporine but negative for the other compounds. CONCLUSIONS An 'all-inclusive' 96-well method for identifying potential drug-drug interactions in vitro was successfully developed and implemented. This is timely, as the recent FDA draft guidance on such studies now recommends using mRNA levels as an important endpoint.