Multiple shoot cultures of two experimental lines of Withania somnifera plants (RS-Selection-1 and RS-Selection-2) were established using nodal segments as explants. The hormonal combinations of benzyl adenine and kinetin not only influenced their morphogenetic response but also differentially modulated the level of biogeneration of withanolide A in the in vitro shoots of the two lines. Interestingly, withanolide-A, that was hardly detectable in the aerial parts of field-grown Withania somnifera (explant source), accumulated considerably in the in vitro shoot cultures of the plant. The productivity of withanolide A in the cultures varied considerably (ca. 10-fold, 0.014 to 0.14 mg per gram fresh weight) with the change in the hormone composition of the culture media as well as genotype used as source of the explant. The shoot culture of RS-Selection-1 raised at 1.00 ppm of BAP and 0.50 ppm of kinetin displayed the highest concentration of withanolide A in the green shoots of 0.238 g per 100 g dry weight tissue. This was a more analytical concentration keeping in view the isolation yields so far reported from the dried roots of the field-grown plant (ca. 0.015 g per 100 g dry weight), even if isolation losses are considered during purification. The enhanced de novo biogenesis of withanolide A in shoot cultures was corroborated with radiolabel incorporation studies using [2-¹⁴C] acetate as a precursor. Production of withaferin A was also found in the in vitro shoot cultures. As this compound is a predominant withanolide of native shoots as well and has been already reported to be accumulated in in vitro shoot cultures, its biogeneration observed in these shoot cultures is not discussed in detail.