Abstract: Separation of enantiomers of atenolol by HPLC was proposed using the Kromasil C18 chromatographic column (250 mm×4.6 mm,5 μm) as stationary phase,and sulfobutylether-β-cyclodextrin (SBE-β-CD in short) as chiral agent added to the mobile phase which is a mixture of 10 mmol·L-1 K2HPO4,methanol and acetonitrile mixed in the ratio of 60+35+5 and containing 20 mmol·L-1 of SBE-β-CD.Acidity of the mobile phase was controlled at pH 8.2.UV detection at the wavelength of 275 nm was adopted in the determination,and column temperature was set at 25 ℃.The flow-rate of mobile phase taken was 0.5 mL·min-1.Volume of sample solution introduced was 20 μL.Under the prescribed conditions,value of separation factor achieved was 1.63.