New, sensitive and rapid chiral HPLC method for enantioseparation and determination of widely used hypnotic drug, zopiclone, was developed. Enantioseparation was achieved on the immobilized amylose based Lux i-Amylose 1 column in polar organic mode in less than 7 min. The effects of the mobile phase composition, type and content of major components as well as acid/base ratio and column temperature on the retention and enantioseparation were investigated. The mobile phase consisted of acetonitril and methanol with small addition of triethylamine and acetic acid with a flow rate of 1 mL min-1. Calibration curves of both zopiclone enantiomers were linear over the concentration range of 5-125 μg mL-1. The limits of detection and quantification for (R)-zopiclone were 5 and 15 ng mL-1 and for (S)-zopiclone were 7 and 21 ng mL-1, respectively. It was demonstrated that the proposed method was selective, precise and robust. Finally, the validated method was applied to the determination of zopiclone enantiomers in the commercial tablets.