Hydrastis canadensis, also known as goldenseal, is among the top 10 selling herbs in the United States, and is used for enhancing general body immunity. Due the increasing demand for wild-crafted goldenseal, plant has been listed in Convention on International Trade in Endangered Species (CITES) Appendix II list as an endangered species. An in vitro propagation protocol for rapidly producing goldenseal plantlets from disk tissue of young leaves was developed. Leaf explants were inoculated on MS medium supplemented with various concentrations of NAA and TDZ for production of callus. Two-month-old calli were sub-cultured on MS media containing cytokinins (BA, Kinetin, TDZ) in different concentrations for shoot initiation. The optimum level of callus induction and maintenance was 5.3 mum NAA in combination with 2.2 muM of TDZ. Shoot multiplication was achieved on MS medium with 2.2 muM TDZ. The alkaloid profile of micropropagated plantlets was similar to the profile of mother plants. These results suggest that our in vitro propagation protocol will produce a positive impact in the conservation of H. canadensis.