A sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of bufuralol enantiomers in plasma and pharmaceutical formulations. Enantiomeric resolution was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with UV detection set at 254 nm. The polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100:0.015:0.010, v/v/v) has been used at a flow rate of 0.5 ml/min. The method is highly specific where other coformulated compounds did not interfere. The stability of bufuralol enantiomers under different degrees of temperature was also studied. The results showed that the drug is stable for at least 7 days at 70 degrees C. The method was validated for its linearity, accuracy, precision and robustness. An experimental design was used during validation to evaluate method robustness. The calibration curves in plasma were linear over the range of 5-500 ng/ml for each enantiomer with detection limit of 2 ng/ml. The mean relative standard deviation (RSD) of the results of within-day precision and accuracy of the drug were <or=10%. There was no significant difference (p>0.05) between inter- and intra-day studies for each enantiomer which confirmed the reproducibility of the assay method. The mean extraction efficiency for S-(-)- and R-(+)-bufuralol from plasma was in the range 97-102% at 15-400 ng/ml level for each enantiomer. The overall recoveries of bufuralol enantiomers from pharmaceutical formulations was in the range 99.6-102.2% with %RSD ranging from 1.06 to 1.16%. The assay method proved to be suitable as chiral quality control for bufuralol formulations by HPLC and for therapeutic drug monitoring.