Recently, ultra performance liquid chromatography (UPLC) has proven to be one of the most promising developments in the area of fast chromatographic separations, as it's been true to the objective of reducing analysis time and maintaining good efficiency. The present report describes development and validation of a new, rapid, and sensitive UPLC method with UV detection to quantify acyclovir in bulk sample using a Waters Acquity HSS T-3 (100 × 2.1 mm, 1.8 µm) column maintained at a temperature of 50 °C with a gradient elution consisting of 1.0% triethyl amine in water and acetonitrile at a flow rate of 0.4 mL/min and detected at 254 nm. Method was found to be selective, linear, accurate, and precise as per ICH guidelines. Detection and quantitation limits of drug were 3 and 8 ng/mL, respectively. Copyright © 2010 John Wiley & Sons, Ltd.