The medicinal plant Plumbago contains a very potent secondary metabolite, plumbagin having many therapeutic properties. Callus culture was induced using explants, leaf, stem and shoot apex, from P. auriculata. Murashige and Skoog media fortified with various growth hormones like NAA, IAA, IBA and 2, 4-D individually and in various combinations were checked for callus induction. Among the growth hormones used, 1 mg/L 2, 4-D showed best callusing. The hormonal combinations of 1 mg/L IAA and 1.5 mg/L NAA in the media exhibited best callus induction using stem internode as an explant. Plumbagin content from root, stem, leaf and callus was analyzed by using thin layer chromatographic technique. The callus derived from stem showed comparable plumbagin content to the in vivo plant parts. Quantitative spectrophotometric analysis of plumbagin from plant samples and callus indicated that plumbagin content was maximum in roots which was followed by callus, stem and leaf samples respectively. Generation of in vitro sources for p!umbagin, for therapeutic applications will serve as a continuous supply and will contribute to preserve the natural plant recourses.