Withania somnifera (Ashwagandha) contains pharmacologically active metabolites such as withaferin A and withanolide A, and it has been considered one of the most important medicinal plants in the Indian Ayurvedic system. However, the commercial demand is too large, and the concentration of these bioactive metabolites in plants is too low. The goal of the current work is to produce these bioactive metabolites more efficiently by using a biotechnological approach. The present chapter focuses on optimizing the techniques for Agrobacterium rhizogenes-mediated transformation of Withania somnifera using different tissues, viz., root, stem, and leaves. Agrobacterium rhizogenes is a great resource for researching gene function, plant-microbe transformation, and increasing secondary metabolite production. In plant biotechnology, A. rhizogenes offers creative approaches to functional genomics and genetic manipulation, making it a promising field. Scientists work to understand the molecular processes underlying the change induced by A. rhizogenes, the bacterium of agricultural benefits.