A direct high-performance liquid chromatographic assay for the determination of labetalol diastereoisomers in plasma without derivatization was developed. Baseline resolution of diastereoisomers was accomplished on a C18 bonded reversed-phase polymeric column with a basic (pH 11.5) mobile phase and isocratic elution. Sample treatment was optimized in order to achieve a complete extraction of labetalol diastereoisomers and to avoid racemization during extraction. Fluorimetric detection improved the selectivity and afforded a detection limit of 3 ng/ml for each diastereoisomer. This method is suitable for routine quantification of labetalol diastereoisomers and has been applied to a pharmacokinetic study in small laboratory animals.