Diabetes showed an escalated incidence in the past recent years in Iran and abroad. This disease is directly correlated with our diet and specially consumption of the normal sugar. The substitution of chemical sweeteners instead of sucrose raised a growing concern over the safety of these compounds. The steviol glycosides from Stevia are the natural non-caloric sweetener with acceptable taste and health properties such as anti-hyperglycaemic, anti-cancerous and anti-hypersensitive. This sweetener can prevent the dental caries with anti-bacterial and anti-fungal properties. Stevia rebaudiana (Bert.) is a perennial herb, belonging to family of Asteraceae. The leaves of Stevia are source of diterpene glycosides, such as stevioside and rebaudiosides, which are estimated to be 200–300 times sweeter than sucrose. The plant originated from the northern regions of South America. It is being commercially cultivated in China, Taiwan, Thailand, Korea, Japan, India and Malaysia. Its medicinal and commercial value led to the urgent demand for large-scale production of Stevia from elite germplasm. The propagation of Stevia using seeds does not led to the production of homogenous population resulting in variability in sweetener level and composition. Vegetative propagation by stem cuttings is also limited by the low number of individuals that can be obtained simultaneously from a single plant. Micropropagation using biotechnological approaches can provide genetically uniform and healthy plants in large scale. So, here we developed a highly efficient method for mass propagation of Stevia rebaudiana (Bert.) using tissue culture technique.