This study aimed to develop a simple, sensitive, and accurate HPLC method for the simultaneous determination of verapamil and norverapamil enantiomers in human plasma. An internal standard (diphenhydramine) was used and separations of verapamil and norverapamil enantiomers were done at room temperature. Chromatographic separation was achieved using a Chiralcel®OD-RH column (5 μm, 4.6 mm i.d. × 15 cm, Daicel Chemical Industries Ltd., Tokyo, Japan) and a mobile phase consisting of 30 mM hexafluorophosphate and acetonitrile (66 : 34, v/v, pH 4.6). Detection of S- and R-verapamil and the metabolites, S- and R-norverapamil was accomplished with a fluorescence detector. The wavelengths of excitation and emission were set at 280 nm and 315 nm respectively.