A simplified, rapid, selective HPLC method for determining five cytochrome P450 (CYP) probe drugs in single run is described. The five specific probe substrates of caffeine, chlorzoxazone, tolbutamide, metoprolol and midazolam, together with the internal standard diazepam, were extracted using liquid-liquid extraction in rat plasma, followed by high-performance liquid chromatography (HPLC) using a C(18) column (5 microm particle size, 250x4.6 mm i.d.). The mobile phase consisted of a methanol and 50 mM phosphate buffer (pH 3.4, 65 : 35). All analytes were separated simultaneously in a single run that lasted less than 22 min. The detection limits range from 0.2-50 microg/ml for caffeine, 0.5-50 microg/ml for tolbutamide, metoprolol and midazolam, 0.2-100 microg/ml for chlorzoxazone, respectively. The intra- and inter-day precisions for five probe substrates were 1.38-11.10% and 3.39-11.33%, respectively, and the accuracy of five probe substrates ranged from 94.92-113.06% and 92.18-112.62%. The limit of quantification (LOQ) was 0.5 microg/ml for tolbutamide, midazolam and metoprolol, 0.2 microg/ml for caffeine and chlorzoxazone. The present method provides a robust, fast analytical tool for the five-probe drug cocktail. Finally, the method was suitable for determining the plasma concentration of these compounds and evaluating the CYP1A2, 2C9, 2D6, 2E1 and 3A4 activities in rats.