A simple, accurate, precise, economical high performance thin layer chromatographic method has been developed for simultaneous estimation of three marker compounds namely stigmasterol in Cissus quadrangularis Linn. extract, ursolic acid in Ocimum sanctum Linn. extract and withaferin A in Withania somnifera Linn. extract present in Uni- Bone herbal formulation. HPTLC separation was performed on aluminium plates precoated with silica gel 60 F254 and toluene-methanol-acetic acid (9: 1: 0.1, v/v/v) as optimized mobile phase. The detection wavelength for simultaneous estimation of three mentioned marker was 520 nm after derivatization with anisaldehyde sulphuric acid reagent. Proposed HPTLC method was validated according to ICH guideline. The retention factor of withaferin A, ursolic acid and stigmasterol were 0.20 ± 0.009, 0.39 ± 0.009, 0.54 ± 0.013 respectively. Linearity for each three markers was obtained in range of 400–2400 ng/band with acceptable correlation coefficient. Good accuracy and precision were obtained as revealed from %RSD value less than 2. In line with the results obtained, a novel, simple, accurate, precise, economical and reliable high performance thin layer chromatographic method was developed for simultaneous estimation of STG, UA and WFA in Uni-Bone herbal formulation
