Abstract: An efficient micropropagation protocol was developed for direct shoot induction from the seedlings of Rubia cordifolia, a species sparsely distributed in the Western Ghats of India. Plant regeneration was achieved using shoot tip, nodal and split nodal half explants on Murashige and Skoog medium. Effect of plant growth regulators like 6-Benzyladenine, Kinetin, 6-Benzyladenine + Indole-3-acetic acid and 6-Benzyladenine + α-Naphthalene acetic acid on shoot multiplication and Indole-3-acetic acid, Indole-3-butyric acid and α-Naphthalene acetic acid on rooting was studied. R. cordifolia shoots showed abundant proliferation and rooting capacity, both of which are significantly influenced by the varying concentrations of the different plant growth regulators. The optimum number of shoot obtained were 5.9 and 5.2 per explants in 2 weeks on the medium supplemented with 1 mg L-1 Benzyladenine and 0.02 mg L-1 Indole-3-acetic acid in nodes and split vertical halves of the node respectively. The results suggest that the nodal explants (intact and split vertical halves) are better sources of shoot formation than the shoot tip explant. Shoot multiplication was rapid and consistent for 4 subcultures with 0.5 mg L-1 Benzyladenine. The best root induction (98%) and survival was achieved on 1 mg L-1 Indole-3-butyric acid followed by 1 mg L-1 Indole-3-acetic acid. Rooted plantlets were successfully transferred to green house conditions with 89% survival. Micropropagated plants displayed normal phenotypes in ex situ conditions. These plantlets can be used to replenish declining populations in the wild, for the extraction of bioactive compounds and reducing pressure on wild stocks.