A new, simple, precise, accurate and robust high‐performance thin‐layer chromatographic (HPTLC) method has been developed for simultaneous estimation of β-sitosterol-D-glucoside, gallic acid, withaferin A and withanolide A in an Ayurvedic formulation, Ashvagandharishta. The plates were developed in mobile phase consisting of toulene: ethyl acetate: formic acid: methanol (6:3:0.1:0.6, v/v/v/v). The retention factors of β-sitosterol-D-glucoside, gallic acid, withaferin A and withanolide A were 0.18 ± 0.01, 0.26 ± 0.02, 0.37 ± 0.02 and 0.57 ± 0.02, respectively. Linearity was in the range of 50‐400 ng/band for β-sitosterol‐D‐glucoside, 150-500 ng/band for gallic acid, 50‐450 ng/band for withaferin A and 50-500 ng/band for withanolide A. Accuracy study showed recoveries of 99.04 - 102.67, 98.77 - 101.96, 99.64 - 102.67 and 100.07 - 101.96 % for β-sitosterol-D-glucoside, gallic acid, withaferin A and withanolide A, respectively. The results obtained in validation assays indicated accuracy and reliability of HPTLC method for simultaneous quantification of β‐sitosterol‐D‐glucoside, gallic acid, withaferin A and withanolide A in Ashvagandharishta. The percent content of β-sitosterol-D-glucoside, gallic acid, withaferin A and withanolide A in Ashvagandharishta were found to be 0.012, 0.064, 0.016 and 0.312 %, respectively.