A rapid, selective and sensitive high performance liquid chromatographic method for the estimation of acyclovir in human plasma has been developed. Chromatography was carried out on a Hypersil BDS C18 column using a mixture of phosphate buffer (pH 2.5) and methanol in a ratio of 95 : 5 v/v as the mobile phase. The eluents were monitored at 254 nm. Lamivudine was used as an internal standard for this study. The retention times for acyclovir and lamivudine were found to be 5.5 and 8.0 min, respectively. The method was found to be linear in the concentration range of 0.201 to 13.545 AgmL-1. The method validated as per FDA guidelines and was found to be suitable for bioequivalence and pharmacokinetic studies.