Abstract: Carboxymethyl-β-cyclodextrin (CM-β-CD) was used as chiral additive for the mobile phase in the resolution of enantiomers of propitocaine hydrochloride by HPLC. The C18 column was used as stationary phase, and a mixture of methanol, acetonitrile and water (mixed in the ratio of 60+15+25) containing 0.4 g·L-1 CM-β-CD was used as mobile phase, at a flow-rate of 0.4 mL·min-1. UV-detection at the wavelength of 202 nm was adopted for the determination, and volume of sample introduced was 10 μL. It was shown that enantiomers of propitocaine hydrochloride were successfully separated by the proposed method and values of separation factor achieved to 1.41.