An isocratic and simple high-performance liquid chromatographic method was developed for the direct resolution of the clenbuterol enantiomers. The method involved the use of a urea type chiral stationary phase (CSP) made of (S)-indoline-2-carboxylic acid and (R)-1-(alpha-napthyl) ethylamine known as the Chirex 3022 column. The stereochemical separation factor (alpha) obtained was 1.27 and the stereochemical resolution factor (Rs) was 4.2 when using a mobile phase composed of hexane:1,2-dichloroethane:ethanol/trifluoroacetic acid (80:10:10 by vol) at 23 degrees C. The (+)-R enantiomer eluted first, with a capacity factor (k'2) of 2.67 followed by (-)-S enantiomer with a capacity factor (k'1) of 3.38. As standard linear calibration curve was constructed over the range of 10 nmol/mL to 250 nmol/mL with a correlation coefficient of 0.999. The method is specific and sensitive with a lower limit of quantitation of 0.1 nmol. Data demonstrating recovery and precision of the assay are presented and the method has been used to monitor and identify quantitatively the profile of the enantiomers of clenbuterol in biological fluids.