The short-acting, benzodiazepine drug midazolam is commonly used as a probesubstrate for drug-drug interaction studies involving cytochrome P450-3A4. The most important enzyme for drug metabolism in humans, CYP-3A4 participates in metabolizing an estimated 52% of drugs. In order to provide bioanalysis for coadministered interaction studies, an HPLC-MS/MS method was developed and validated to quantify midazolam and its major metabolite α-hydroxymidazolam in human plasma. The validated method was then applied to analyze clinical pharmacokinetic study samples.