Stevia rebaudiana Bertoni, a natural non-caloric sweetener, can be used as sugar substitute for diabetic patients. Conventionally, it is propagated by seeds or stem cuttings which has a limitation of quality and quantity of the seed material. In this study a protocol for shoot regeneration of Stevia from nodal segments, callus formation from leaf explants and plant regeneration from callus were investigated. The results showed that Murashige and Skoog (MS) medium containing vitamins supplemented with 0.2mg/l NAA and 0.15mg/l BAP was found to be an efficient medium for not only shooting with 100% of multiple shoot proliferation from nodal segments but also callusing with 92.6% of nodular callus induction from leaf explants, however the callus failed to differentiate into shoots. The highest rooting was obtained (83.33%) in MS medium supplemented with 0.5mg/l IAA. The rooting plantlets were successfully acclimatized in the mixture of 1 soil: 1 sand: 1 decomposed rice straw (v/v) with 100 per cent survival rate after 3 weeks and no observable morphological abnormalities. This developed protocol could efficiently be used for large scale production of Stevia rebaudiana Bertoni through in vitro propagation.