Withania somnifera (L.) Dunal is an important medicinal plant with many pharmaceutical and therapeutic uses ranging from immunomodulation to anticancer property. Field cultivation of W. somnifera is time consuming and it not able to meet current global demands. For commercial production of withanolides, field grown plant materials have generally been used but the quality and uniformity in the levels of active constituents are highly affected by genotype and environmental conditions. In vitro culture methods are the best alternative and have possible ways to enhance commercial production of withanolides. In this review, we summarize our recent findings on characterization and screening of wild accessions of W. somnifera collected from Tamil Nadu state. Further, an improved Agrobacterium rhizogenes mediated hairy root culture system was developed and this system providing an efficient tool attaining better transformation efficiency and useful for commercial in vitro production of withanolides.