Chitrak (Plumbago zeylanica L.), a renowned traditional medicinal plant, is being exploited extensively for its roots which are employed in the preparations of many important herbal products (e.g. Dashmularisht, Chitrakadivati) possessing anticancer, anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective properties. P. zeylanica roots, being the major source of plumbagin, were used for its isolation. Plumbagin isolation from in vivo roots was done employing column chromatography and thin layer chromatography (TLC) using a hexane: ethyl acetate (70:30) mobile phase. A total of 65 fractions were obtained which pooled down to 8 (F1–F8) based on their similar Rf values. Of the 8 fractions, F2 gave a single spot corresponding to that of the standard plumbagin which subsequently was validated and confirmed through ¹H-NMR and FT-IR. For elicitation to increase plumbagin, in vitro root callus initially raised on Murashige and Skoog medium +5 μM TDZ was augmented with different concentrations of precursors (sodium acetate, l-tyrosine), biotic (chitosan, proline, lysine, salicylic acid, yeast extract) and abiotic [Pb(NO₃)₂, CdCl₂] elicitors either alone or in combinations. The optimum increase of up to 2.07 and 2.64–folds in plumbagin was seen when callus cultures were fed individually with sodium acetate (1 mg/L) and l-tyrosine (25 mg/L), respectively. Similarly, chitosan and yeast extract when used alone, the plumbagin content was enhanced 4.58–fold at 50 mg/L and 6.50–fold at 100 mg/L, respectively. However, an enormous increase of 12.08–fold plumbagin content occurred when a combination of 50 mg/L chitosan + 100 mg/L yeast extract was used. A combination of salicylic acid (25 μM) and yeast extract (100 mg/L) also enhanced plumbagin content up to 8.23–fold. Thus, this is our first report of scaling up to 12.08–fold plumbagin content using simple and cost effective elicitors.