Cromakalim is an anti hypertensive drug (potassium channel activator) that acts as potent vasodilator functioning by hyperpolarization of vascular smooth muscle membranes and opening of potassium channels. Attempts have been made to resolve the enantiomers of cromakalim (3S,4S and 3R,4R) on polysaccharide chiral stationary phases (CSPs) under reversed-phase mode. The columns used were Chiralpak AD-R, Chiralcel OD-R, and Chiralcel OJ-R. The mobile phases used were water-acetonitrile (70: 30, v/v) for the Chiralpak AD-R and the Chiralcel OD-R columns, and water-acetonitrile (80: 20, v/v) for the Chiralcel OJ-R column. The flow rate of the mobile phases was 0.5 mL/min. The detection was carried out at 220 nm. The alpha-values of the resolved enantiomers were 1.26, 1.23, and 1.13 on Chiralpak AD-R, Chiralcel OD-R, and Chiralcel OJ-R columns, respectively, while the values of R, were 1.20, 0.40, and 0.30 on Chiralpak AD-R, Chiralcel OD-R, and Chiralcel OJ-R columns, respectively. Concentration of cromakalim in human plasma was determined by using solid-phase extraction method.