A sensitive, accurate and rapid reverse phase Liquid chromatography/tandem mass spectrometry (LCMS/MS) method was described to estimate acyclovir in human plasma. Detection was made at m/z 226.1/152.2 for Acyclovir and 346.1/198.1 for internal standard using ESI Positive ion spray ionization mode. Analyst 1.5.1 software was used for the quantification. The stationary phase was Ascentis C18, 50 X 4.6 mm, 5μm column. The separation method developed produce recovery of 84.58%. Acceptable intra-day and inter-day precision (<15%) and accuracy (<10% diff.) were observed over the linear range of 82.967 to 4006.948 ng/mL. The absence of any matrix effects was displayed. The retention time of analyte and internal standard was 1.05 and 1.24 minutes. The developed and validated method was successfully applied for bioequivalence studies.