A sensitive enantioselective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of plasma concentrations of (−)( R)- and (+)( S)-chlorpheniramine (CP) and their metabolites, desmethyl-chlorpheniramine (DCP), didesmethyl-chorpheniramine (DDCP) and chlorpheniramine N-oxide (CPNO). Enantioselective separations were achieved on a β-cyclodextrin chiral stationary phase (CYCLOBOND I 2000™) with a mobile phase consisting of diethylamine acetate (0.25%, pH 4.4):methanol:acetonitrile {85:7.5:7.5, (v/v/v)} and a flow-rate of 0.5 ml/min. For CP, the enantioselectivity ( α) of the separation was 1.12 with a resolution factor ( R s) of 1.17. The method was validated for CP by using mass spectroscopy detection (MSD). Concentrations of each enantiomer could be measured down to 125 pg/ml from a 1-ml plasma sample. Extracted calibration curves were linear from 0.13 to 50.00 ng/ml for each enantiomer. The method was applied to samples from two clinical studies.