A protocol for in vitro production and acclimatization of wrightia tinctoria (rox.) R. Br., is reported. Under the regime used in the study, the most critical factor for rooting was activated charcoal (0.05%) along with 6 μM Naphthalene aceticacid. The plantlets hardened with longer photoperiods had better survival percentages when compared with those acclimatised over shorter photoperiods. Hardened plantlets obtained within 45 days, and replanted with 86.8 % survival. This in vitro method can be used for rapid mass clonal propagation of W. tinctoria for conservation strategies, commercial production irrespective of season and gene transformation studies for the production of phytomedicines.