Objective: Xanthium strumarium is a cocklebur or burweed belonging to family Asteraceae and commonly found as a weed, is widely distributed in North America, Brazil, China, Malasia, and hotter part of India. The herb is traditionally used mostly in treating several ailments. The present study deals with development and validation of a reliable reverse phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Stigmasterol and β-sitosterol in the various extracts of the plant. Methods:The proposed method utilizes a Qualisil Gold C18 column (250×4.6 mm), 5 µm particle size, under isocratic elution conditions with the mixture of acetonitrile:ethanol:water (85:14:1 v/v/v) at 25° as a mobile phase. An effluent flow rate of 1 ml/minute and ultraviolet detection at 202 nm was used for the analysis of Stigmasterol and β-sitosterol. Results:The described method was linear in the range of range of 100-500 µg/ml and 10-500 µg/ml for stigmasterol and β-sitosterol respectively, with excellent correlation coefficients. The precision, robustness, and ruggedness values were also within the prescribed limits (<2%). The recovery values were within the range, which indicates that the accuracy of the analysis was good and that the interference of the matrix with the recovery of phytosterols was low. The phytosterols were found to be stable in a stock solution for 24 hrs (percentage relative standard deviation was below 2%) and no interfering extra peaks were observed under controlled stress conditions. Conclusion:The proposed method is simple, specific, precise, accurate, and reproducible and thus can be used as appropriate method for routine analysis of X. strumarium phytosterols in quality control laboratories.