Pravastatin, a natural bioactive compound inhibits cholesterol biosynthesis which is isolated from fungus by fermentation process. A rapid, sensitive and robust novel reversed phase-HPLC method with UV detection set at 238 nm was developed and validated for quantification of pravastatin in pure drug form and pharmaceutical formulation (Pravachol). A liquid-liquid extraction technique was applied for the preparation samples and chromatographic separation was performed on Symmetry® C18 (5μm, 3.9 × 150 mm) column with a retention time of 1.95 min using isocratic elution of mobile phase. The mobile phase used in this work consisted of a mixture of methanol and phosphate buffer (70:30%, v/v, pH 4) which was delivered at a flow rate at 1.5 ml/min. Developed HPLC-UV method was validated for linearity (R2 = 0.999), accuracy, precision, sensitivity, system suitability, selectivity, and robustness. Box-Behnken experimental design was applied in the validation of robustness. Three independent variables such as the composition of mobile phase, flow rate and pH of mobile phase were investigated. The results indicated that a slight change in the mobile phase composition and flow rate affects the responses, while pH had no significant effect on the response. Overall, the proposed HPLC-UV method was found to be simple, sensitive, and highly robust for routine analysis of pravastatin.