The aim this study is to obtain an effective and efficient of method for assay of acyclovir cream determination. Chromatographic separation on a reverse phase a YMC - Triart C18 column (4.6 x 150 mm, 5 μm), the mobile phase consisted of water and acetonitrile (95: 5 v/v) at a flow rate 1.2 mL/min with UV-Vis detection at 252 nm, volume injected was 20 μL, with UV detection at 252.0 nm. The retention time of acyclovir was ± 3.5 min. Validation studies were achieved by using the limit of detection (LOD) and limit of quantification (LOQ), precision, accuracy, selectivity parameter. The LOD and LOQ values were 0.27 µg/mL and 0.83 µg/mL. The average standard deviation (RSD) value of precision is 0.35% and accuracy is 0.34%. Robustness shows that the method is resistant to variation in the mobile phase, the Acyclovir cream analysis method meets the ICH 1994 requirements, so it can be applied to the assay of Acyclovir determination in Cream dosage forms.