Protocol for micropropagation of a known medicinal plant Vinca (Catharanthus roseus (L.) G. Don) from axenic explants had been standardized. Internodes and shoot tips were cultured on MS medium containing a varied concentration of IAA, 2-4 D, NAA, BAP and Kinetin in combination were used. The best combination of growth regulators for callus induction was at 1 mg L-1 kinetin with 0.9 mg L-1 of IAA, closely followed by 1.0 mg L-1 of kinetin with 1.0 mg L-1 of IAA for internodes as well as shoot tip. While for internodal culture 0.5 mg L-1 of 2,4-D and 1.5 mg L-1 BAP was optimum. A method of alkaloid extraction and assay of secondary metabolite in callus culture was accomplished. The HPLC assays method quantified major compounds of the secondary metabolism of callus cultures of C. roseus.