Chlorophytum borivilianum Sant. et Fernand an endangered herb is valued for several medicinal properties in its tuberous roots. An efficient and reproducible method for inducing in vitro tubers from stem disc explant has been developed. Stem disc possessing shoot buds were induced to develop multiple shoots in Murashige and Skoog (MS) medium supplemented with vitamins, 3% sucrose, 0.8% agar and 5 mg/L benzylaminopurine (BAP). Healthy regenerated shoots were rooted in MS basal medium containing 3% sucrose (w/v), 0.8% agar supplemented with indole-3-acetic acid (2 mg/L). On further sub culturing, the maximum percentage of tuber formation was obtained in growth hormone free half (½) MS liquid media supplemented with vitamins and 1.5% sucrose after 8 to 9 weeks. The saponin contents of the in vitro and in vivo raised tubers were qualitatively and quantitatively analyzed by highperformance liquid chromatography (HPLC) and liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). There was a significant similarity in the saponins in both tubers. The in vitro raised tubers showed similar high metabolite content than in vivo grown tubers which is required for medicinal applications. The rooted plantlets were transferred to peat and sand (2:1) with more than 80% success. This is the 1st report of in vitro tuber formation and secondary metabolites screening of C. borivilianum. This work will give a strong impetus to the pharmaceutical and neutraceutical sectors.Keywords: Chlorophytum borivilianum, tuber formation, saponin, liquid medium, high-performance liquid chromatography (HPLC)African Journal of Biotechnology Vol. 12(20), pp. 2900-2907