Stevia rebaudiana Bertoni, is a perennial herb belongs to the Asteraceae family. The plant has strong sweet taste and very few calories. It is estimated to be 300 times sweeter than sugar cane. Seed germination is notably very poor due to infertile small size seeds and their self incompatibility and lower number of vegetative cutting. It is a newly introduced plant in Egypt, So, the present investigation was carried out to standardize a protocol for in vitro masspropagation and acclimatization of Stevia rebaudiana plants. In establishment stage MS medium supplemented with 0.25mg/l BAP or 0.25mg/l Kn in combination with PP₃₃₃ enhanced shoot number significantly (10.00 and 5.50 shoots/explant, respectively). In multiplication stage, adding growth retardant (PP₃₃₃, CCC and PEG) as well as AC positively affected growth parameters. In rooting and acclimatization stages auxins especially IAA and NAA played an important role in combination with PP₃₃₃ and AC.