Abstract Solanum xanthocarpum is an important medicinal plant belonging to the family Solanaceae. A simple, rapid, sensitive, accurate, repeatable and robust HPTLC method has been developed and validated for the estimation of β- sitosterol in the leaf extract of Solanum xanthocarpum. β- sitosterol was estimated at 206 nm by densitometry using Silica gel 60 F254 as stationary phase and Toluene: Ethyl acetate (7:3), and as mobile phase. Linearity was observed in the concentration range 20-120ng/spot for β-sitosterol. The limit of detection and limit of quantification were found to be 0.07 & 8.17ng/spot respectively for β-sitosterol. Developed method was validated according to the ICH guidelines with respect to precision, accuracy, specificity and robustness. The technique has been applied for the first time for the estimation of β-sitosterol in fruit extracts of Solanum xanthocarpum. Statical analysis data indicate the accuracy and reliability of the method. Keywords Solanum xanthocarpum; β-sitosterol; Herbal analysis Validation, Kantakari, ICHguidelines