Multiple shoot cultures of Withania somnifera were established from single shoot tip explants and their potential for the production of two principle withanolides, withaferin A and withanolide D was investigated. Shoot tips grown on MS medium supplemented with BA (1 mg I-1) induced 10.0 ± 1.15 microshoots per explants and shoot cultures accumulated both withanolides (withaferin A = 0.04%, withanolide D = 0.06%). Supplementation of MSSM (solid) agar medium with 4% sucrose enhanced accumulation of both withaferin A (0.16%) and withanolide D (0.08%). Reduction of the agar concentration to 0.16% increased the number of microshoots induced per explant to 25.5. MSSM liquid medium containing 10% coconut milk favoured a maximum increase in biomass (27 fold); number of microshoots induced (37.6 ± 1.45) as well as accumulation of withaferin A (0.14%).