Stevia rebaudiana is considered a valuable natural sweetener and medicinal plant containing sweetener compounds in its leaves. Nowadays, plant drugs are frequently considered to be free from side effects and less toxic than the synthetic one, for that people and some pharmacological companies are heading to the herbal medicine. Thus, to meet the increased demand for raw material of this medicinal herb, I described in this study an efficient regeneration protocol using shoot tip and nodal explants and different PGRs on MS medium for healthy biomass production. The optimum shoot growth with a high number without callus formation was obtained using MS medium in the presence of 1.0 mgL-1 BA after 6 weeks which shows (4.06-4.46) shoot buds/explant after 6 weeks. The combination of 1.0 mg L-1 BA with 0.5 mg L-1 IBA was found to be best combination for shoot multiplication that recorded 6.66 shoots/explant with 4.46 cm shoot length. Healthy elongated shoots were harvested and then cultured on half strength MS basal salts medium without vitamins medium in the presence of different concentrations of IBA and NAA individual or in combination with IAA or 2,4-D. Moreover, the optimum root growth with high roots number (4.46) with (76.6 %) root formation was observed using 1.0 mg L-1 IBA on half strength MS medium. Therefore, well-developed healthy in vitro rooted plantlets after 6 weeks were successfully acclimatized and they grew as normal plants in the greenhouse.