Introduction: Chiral HPLC is a chromatographic separation technique used to separate the Enantiomer or Diastereomer from the main active compound. Aim: A simple, rapid and robust reverse phase chiral HPLC method was developed and validated for the quantification of Lamivudine Enantiomer in Lamivudine drug substance. Methodology: The Enantiomer of Lamivudine was resolved in Lux cellulose-5 column using a mobile phase containing Methanol: Diethylamine (100:0.1 v/v) at a flow rate of 0.5 mL/min. Results: The resolution between Enantiomer and Lamivudine was found to be more than 1.5 in an optimized method. The developed method was validated and proved that the method was specific, sensitive, accurate and precise as per ICH. The limit of detection and limit of quantification for Lamivudine Enantiomer was 0.004% and 0.012% respectively. The linearity curve was found to be linear and the correlation coefficient obtained was 0.999. The average percentage recoveries of Enantiomer were in the range of 97 to 102%. Conclusion: The proposed method was found to be suitable and accurate for the quantitative determination of Enantiomer in Lamivudine drug substance.