Background: The metabolic activity of P450 enzymes in vivo can be determined using selective probe drugs. The simultaneous administration of multiple CYP-specific probe drugs is commonly known as the cocktail approach. Disadvantages of a cocktail are large volumes of samples required for analysis and timeconsuming analyses. The aim of this study was to develop and validate a simplified but sensitive method for the simultaneous quantification of 5 probe drugs [caffeine (CYP1A2), metoprolol (CYP2D6), midazolam (CYP3A4), omeprazole (CYP2C19), and S-warfarin (CYP2C9)] in a previously validated cocktail using a liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method. Methods: The method entailed a single method for sample preparation that enables quick processing of the samples containing all 5 probe drugs in a small volume of blood (10 mL) followed by a chiral and nonchiral LC-MS/MS method. The method was validated for selectivity, specificity, resolution of racemic warfarin, linearity, accuracy, imprecision, recovery, process efficiency, ionization efficiency, and carryover effect. Results: The method showed good selectivity without matrix interferences and differentiated S-and R-warfarin enantiomers with adequate resolution (Rs = 1.55). For all analytes, the mean process efficiency was .95%, and the mean ionization efficiency was .97%. Furthermore, the accuracy was between 94.9% and 108% for all analytes, and the within-and betweenrun imprecision were ,11.7% for the lower limit of quantification and ,12.6% for the middle level and upper limit of quantification. Conclusions: The method presented here enables the simultaneous quantification of the 5 probes in a very small blood volume (10 mL). Furthermore, it is less time consuming than previously reported methods because it requires only 1 simple method for sample preparation followed by a nonchiral and chiral LC-MS/MS method that can be performed sequentially.