Moringa oleifera is a highly valued medicinal plant. The present research reports callus cultures of M. oleifera Lam., established from seeds and nodal segments on Murashige and Skoog’s (MS) medium using different concentrations and combinations of auxins and cytokinins. Best induction of callus was observed at BAP:IBA (3 mg l⁻¹ each). Shooting and rooting from callus in terms of morphogenesis were observed in MS media supplemented with BAP:KN (2:0.2 mg l⁻¹) and IBA:NAA (3:0.5 mg l⁻¹), respectively. Multiple shooting was observed at treatment dose of BAP:NAA:IAA (1:1:0.2 mg l⁻¹). Regenerated shoots were rooted and mature plants were established, acclimatized, and thrived in greenhouse conditions. Over 95 % of plantlets survived after transplanting plantlets into trays with a mixture of sand and perlite (2:1) for 20 days. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of bioactive constituents of this medicinal plant. Further qualitative and quantitative production of steroidal sapogenins (diosgenin and tigogenin) from various morphogenetic stages was studied using TLC, PTLC, IR spectra, HPLC and GC–MS analysis. Steroidal sapogenins were maximum in the callus associated with rooting. Various stages were further analyzed for their antioxidant potential.