Withania somnifera (L.) Dunal commonly known as Ashwagandha, isan important medicinal herb belonging to the solanaceae family. It is rich source of withanolide A, withanolide D, withaferin A, and withanone. Presence of various pharmacological properties, the plant has huge demand in pharmaceutical industry due to its pharmacological properties. However, there is large gap between demand and supply, thus, it is necessary to develop in vitro propagation techniques for this medicinally valuable plant. In the present research in vitro propagation of, Withania somnifera, direct regeneration of nodal explants and their multiplication have been optimized using cytokinin BAP and Kin (2.0 μm/l). MS media with nodal explants supplemented with BAP (2.0μm /l) produced maximum average number of shoots (5±0.53) and average shoot length was found to be 4.8±0.28 cm. Shoot proliferation was observed on BAP(4.0μM)+ Kinetin (4.0μM) with maximum number of shoots 8.03 ± 0.13. Elongated shoots then sub cultured for root induction on MS media supplemented with IBA, IAA and NAA (2.0-8.0μm/l) Maximum average number (6± 0.10) and average length 7.6±0.18cm of roots were observed on MS media supplemented with IAA (2.0μm/l).The rooted plantlets when transferred to poly bags in the greenhouse showed 85% survival frequency. This protocol useful for production of large quantity withaferin and conservation of this valuable medicinal plant