An enantioselective high-performance liquid chromatographic method for determination of enantiomers of mosapride citrate in bulk drugs and pharmaceuticals using UV–vis and polarimetric detectors in series has been developed. Baseline separation with resolution >2.0 was achieved on a column containing amylose tris-(3,5-dimethylphenylcarbamate) as stationary phase using a mobile phase consisting of n-hexane:ethanol:triethylamine (80:20:0.3, v/v/v) at 40 °C. The detection was carried out at UV-276 nm and enantiomers were identified by polarimetric detector. The effect of ethanol, 2-propanol, TEA, temperature and mobile phase flow rate on separation of MSP enantiomers was studied and the method was validated with respect to accuracy, precision, linearity and limits of detection and quantification. The linearity of the method was studied between 6.25 and 50 μg/ml and r 2 was >0.9997. The recoveries were in the range 99.63–100.22%, the method was suitable not only for process development of mosapride citrate but also for quality assurance of the individual enantiomers in bulk drugs and pharmaceuticals.