Abstract: Withania somnifera (L.) Dunal., one of the 32 prioritized medicinal plants of India, is well known for its importance in the Ayurveda system of medicine. Attempt was made to establish an efficient plant regeneration protocol for a commercial cultivar 'Poshita' and to acclimatize the plantlets ex vitro, so as to reduce the cost. Results revealed that shoot induction was possible only after an intervening callus phase, irrespective of the concentration of growth regulators present in the nutrient pool. Nodal explant cultured on MS media supplemented with 1 mg L^-1 BAP + 0.5 mg L^-1 NAA showed superiority in callus induction capacity over epicotyls and leaves. Nodal segments when cultured on a media containing BAP alone could induce shoots in cent per cent explants. Highest number of shoots (5.8) was obtained in media containing 2 mg L^-1 BAP. Number of adventitious buds was found to be maximum (13) with epicotyl explant and 1 mg L^-1 BAP combination. Nodal explants cultured on high concentration of BAP (4 mg L^-1) showed highest incidence of malformed shoots (4.3). A total of 66.7 % plantlets could root and establish ex vitro even without auxin treatment and survival rate increased (87.5%) with increase in IBA concentration to 500 mg L^-1. The present protocol can be exploited on a commercial scale to obtain maximum benefits from the improved cultivar. Furthermore, ex vitro hardening can help to reduce the cost of production and thereby make the tissue culture industry more profitable.