A simple isocratic RP-HPLC method has been developed and subsequently validated for the determination of Lamivudine in API form and pharmaceutical dosage forms as per ICH guidelines. The separation achieved on a reversed phase Symmetry ODS (C18) RP Column, 250 mm x 4.6 mm, 5µm as a stationary phase and Phosphate Buffer and Methanol in the ratio of 46:54 (pH-3.2) as mobile phase at a flow rate of 1.0 ml/min. The UV detection was performed at 206 nm. The retention time for Lamivudine was found to be 3.622min. The detector response was linear in the concentration range of 60-140 μg/ml. The respective linear regression equation being Y= 48313.x + 71968 with R2 = 0.9993. The percentage of Lamivudine in pharmaceutical dosage form was found to be within in the limits. The limit of detection and the limit of quantification were found to be 0.08μg/ml and 0.24μg/ml respectively. The results of the study showed that, the proposed RP-HPLC method was simple, rapid, precise, accurate and specific, which can be used for the routine determination of Lamivudine in API form and pharmaceutical dosage form.