The method for the separation of clorprenaline hydrochloride enantiomers was developed using reversed phase high-performance liquid chromatography(RP-HPLC)with carboxymethyl-β-cyclodextrin(CM-β-CD)as chiral mobile phase additive.The effects of concentration of CM-β-CD,organic solvents(type and concentration)and flow rate on the enantiomers separation was studied systematically.Separation was performed on a Diamond C18 column.The best separation was obtained with methanol-acetonitrile-water(80∶10∶10,v/v/v)mixed with 0.40g/L CM-β-CD as mobile.The optimal flow rate was 0.20 mL/min.The detection wavelength was set at 216 nm.Under the optimized conditions,the resolution of enantiomers was 1.60 in 30 minutes,The method was simple and fast with small relative standard deviations.