Abstract: A method of HPLC was proposed for the resolution of enantiomers of ethyl 3-hydroxybutyrate using Chiralcel OD-H chiral column (4.6 mm×250 mm, 5 μm) for separation. A mixture of n-hexane and isopropyl alcohol (100+5) was used as mobile phase with flow-rate of 1.0 mL·min-1. Column temperature of 25 ℃ was chosen. Under the optimized conditions, enantiomers of ethyl 3-hydroxybutyrate were successfully separated within 11 min, with resolution of 4.25.