The resolution of clenbuterol hydrochloride enantiomers was achieved by thin-layer chromatography on silica gel GF254 plates impregnated w ith beta-cyclodextrin. The effect of stereoselective auxiliary (acetonitrile and alcohol) was investigated. Resolution of clenbuterol hydrochloride enantiomers could be attained by using alcohols of butanol, 2-butanol or tert-butanol together with acetonitrile as developing solvent. The optimal conditions of resolution were determined as follows: a plate prepared with 15.00 g silica gel GF254 impregnated with 1.00 g beta-cyclodextrin, acetonitrile-2-butanol (20:80, v/v) as developing solvent and developed at room temperature. Under these conditions, Rf of the two isomers of clenbuterol hydrochloride enantiomers were 0.34 and 0.72 respectively. The resolution was 4.09 with baseline separation and the spots in chromatogram were almost of the same size.