Holostemma ada-kodien is a medicinal plant belongs to the family Asclepiadaceae. Root tubers contain alpha amyrin, Lupeol, and β- Sitosterol. The present study was to standardize in vitro culture of Holostemma ada-kodien Schult. On Murashige and Skoog (MS) medium supplemented with different combinations of growth regulators to induce shoot, root and callus formation. Among several combinations of BAP, NAA and Kinetin studied, nutrient medium supplemented with 0.1mg/l BAP and 1.0 mg/l Kinetin induced maximum number of shoots. Highest callus proliferation was observed in the medium supplemented with 1.0 mg/l BAP and 1.0 mg/l NAA. Root initiation was also observed in the same medium.