Oldenlandia biflora L. (Rubiaceae) is a small, straggling, delicate plant appearing in the rainy season on the banks of water courses. The whole plant is used in medicine. An efficient protocol for rapid multiplication of O. biflora was standardized in this study. The leaf explants were cultured on MS medium supplemented with different concentrations of IAA, IBA, NAA and 2,4-D for callus induction. All these auxins produced calli with a little variation in callus morphology. In combination with low concentration of BAP, green compact calli were developed. Shoots were initiated from the calli on MS medium containing cytokinins and low concentration of auxins. Of the two cytokinins, BAP was found to produce more number shoots when compared to KIN. In combination of these two cytokinins, number of shoots were increased. More than 40 shoots were obtained on MS medium containing 6 µM BAP+2 µM KIN+2 µM NAA. Basal medium alone is enough to initiate roots in this plant. However, the addition of auxins enhanced the rooting. The well rooted plantlets were transferred to root trainer for hardening. Subsequently these plantlets were established in the field through acclimatization. The highest rates of hardening and acclimatization were observed in this study with 95% and 90% respectively.