A fast and robust LC/MS-based cytochrome P450 (CYP) inhibition assay, using human liver microsomes, has been fully developed and validated for the major human liver CYPs. Probe substrates were phenacetin, diclofenac, S-mephenytoin, and dextromethorphan for CYP1A2, CYP2C9, CYP2C19, and CYP2D6, respectively. Midazolam and testosterone were chosen for CYP3A4. Furafylline, sulfaphenazole, tranylcypromine, quinidine, and ketoconazole were identified as positive control inhibitors for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. To increase the throughput of the assay, a one-point method was developed, using data from CYP inhibition assays conducted at one concentration (i.e., 10 mM), to estimate the drug concentration at which the metabolism of the CYP probe substrate was reduced by 50% (IC 50 ). The IC 50 values from the one-point assay were validated by correlating the results with IC 50 values that were obtained with a traditional eight-point concentrationresponse curve. Good correlation was achieved with the slopes of the trendlines between 0.95 and 1.02 and with R 2 between 0.77 and 1.0. Throughput was increased twofold by using a Cohesive multiplexing high-performance liquid chromatography system. The one-point IC 50 estimate is useful for initial compound screening, while the full concentration-response IC 50 method provides detailed CYP inhibition data for later stages of drug development.