Cytochrome P450s (CYPs) play a major role in drug detoxification, and inhibition of CYP-mediated metabolism may lead to accumulation of toxic drug levels in the plasma. To prevent adverse drug-drug interactions, new drug candidates are routinely tested for their ability to inhibit these enzymes. This unit describes a variety of protocols for evaluating new chemical entities as inhibitors of CYP activity. An example protocol illustrates a high-throughput screening format using a fluorogenic probe, and a method for evaluating a test compound as a time-dependent inhibitor of CYP is also described.