Stevia plants (Stevia rebaudiana Bertoni) became an economically important medicinal plant act as a sugar substitute for diabetic and obese people in many countries. In Egypt, we faced a shortage in sugar production and no more available water for irrigation of sugarcane or sugar beet crops. Stevia, a non-caloric sweetener, is being many times sweeter than sucrose also is required less water. Stevia has difficulties in propagation and improvement through seeds and vegetative methods, so in vitro biotechnology is the protocol has been an efficient alternative for propagation and improvement this plant. Leaf segments as the best explants were used in the present work to in vitro culture of stevia. For surface sterilization, the best exposure time and the combination were found that mercuric chloride )0.1%( for 2 minutes, ethanol )70%( for a one minute, and sodium hypochlorite solution (commercial Clorox®) (15%( for 15 min. Murashige and Skoog (MS) media supplemented with a wide range of concentration and combination of plant growth regulators (PGR) were tested. MS medium containing 1.0 mg/l 2, 4-D (2,4 Dichlorophenoxy acetic acid) + 1.0 mg/l NAA (Naphthalene acetic acid) was the highest and significant callus induction percentage (96℅). However, the narrow range was observed for plant regeneration (1.8 – 3.6 plantlets/callus) as well as plantlet length (1.176 – 3.370 cm). A significant variation was observed for leaves number/regenerated plantlet (3.20 to 6.14). MS medium contains 1.8 mg /l BAP (Benzyl amino purine) + 0.12 mg /l NAA resulted the highest regenerated plantlets/callus (3.600). Healthy rooted plants were obtained and transferred to pots for ex vitro hardiness under controlled environmental conditions. The effects of callus induction medium on the activity of mitotic cell division and their mitotic index were evaluated. Wide range and significant variation were observed among mitotic phases and mitotic indexes (MI) grown on different tested MS media. Stevia calli were grown on MS medium + 2.0 mg/l NAA exhibited the highest MI value (9.82%) with a significant difference with all other six used media. Calli cells at metaphase were showed the normal chromosome number 2n=22.