In a continuation of earlier findings on glycoalkaloids and steroids in different plant parts of Solanum jasminoides Paxt., this study reports the isolation and identification of diosgenin and solasodine in its callus culture. Callus tissue cultures were established from sterilized stem pieces with buds on Revised Murashige and Skoog's medium (RT) supplemented with 1 ppm 2,4-dichlorophenoxyacetic acid (2,4-D) and 15 agar, maintained for 14 months by subculturing every 6-8 weeks, then transferred to fresh RT medium and harvested at 2, 4, 6, and 8 weeks for analysis. Growth index (GI) was calculated, and steroidal sapogenins and glycoalkaloids were analyzed via hydrolysis, extraction, thin-layer chromatography (TLC), preparative TLC, crystallization, and spectral studies. Results showed steady growth index increase up to 6 weeks (maximum 7.0) then decrease. Diosgenin concentration was maximum at 8 weeks (0.099%), solasodine at 6 weeks (0.19%). Compared to intact plants (higher metabolite amounts in roots, flowers, aerial parts), callus cultures had lower amounts but could biosynthesize diosgenin (absent in stem pieces) from stem-derived callus. Thus, the cell culture system of S. jasminoides has an advantage in synthesizing these metabolites with higher potential despite lower amounts than intact plants.