Abstract A new, accurate, rapid and effective isocratic reverse-phase high performance liquid chromatographic (RP-HPLC) method was developed, optimized and validated for the estimation of Lamivudine (LAM) and Zidovudine (ZID) in bulk and pharmaceutical dosage forms. The drugs were estimated using Shiseido C18 (250 mm x 4.6 mm, 5 µm particle size) column. A mobile phase composed of phosphate buffer (pH 3) and ACN in the ratio of 70:30, v/v at a flow rate of 1.0 ml/min was used for the separation. Detection was carried out at 228 nm. The linearity range used was 7.5-22.5µg/ml for LAM and 15-45µg/ml for ZID with retention times (Rt) of 2.512 min and 3.721 min for LAM and ZID respectively. The correlation coefficient values were found to be 0.999. Precession studies showed %RSD values less than 2% for both the drugs in all the selected concentrations. The percentage recoveries of LAM and ZID were found to be 99.06% and 99.88% respectively. The assay results of LAM and ZID were within the limits of 98-102%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.1ng/ml and 0.1µg/ml for LAM and 0.1µg/ml and 0.5µg/ml for ZID respectively. The method was validated as per the International Conference on Harmonization (ICH) guidelines. The developed method was successfully used for the quantitative analysis of commercially available dosage form.