A UV-spectrophotometric method for simultaneous estimation of Acyclovir and Silymarin in niosome formulation has been developed. Methanol is used as solvent for preparation of stock solution and further dilutions are prepared in distilled water. Acyclovir and Silymarin exhibit absorption maxima at 251nm and 287nm respectively. The system obeyed Beer's law over the concentration range of 2-10 μg/ml and 10-50 μg/ml for Acyclovir and Silymarin respectively. Method was validated for linearity, range, accuracy, precision and recovery studies as per ICH norms. Thus the proposed method was found to be rapid, specific, precise, accurate and cost effective quality control tool for the routine analysis of Acyclovir and Silymarin in niosome formulation.