Synergism between various plant growth regulators is reported to be a key factor for the development of efficient in vitro propagation for any plant. Therefore, the current study examines the synergistic impact of plant growth regulators on in vitro propagation of Eclipta alba, an important medicinal plant possessing diverse medicinal properties. For the establishment of aseptic cultures, nodal segments were employed as explants on MS medium supplemented with 2.5 μM of 6-benzyle adenine (BA). Varying concentrations of BA and Kinetin (KIN)(0.0-5.0 μM), either alone or in combination with α- naphthalene acetic acid (NAA @ 0.0-5.0 μM) and indole 3-acetic acid (IAA@ 0.0-5.0 μM), were found to be effective for promoting shoot proliferation. Compared to KIN, BA was found to promote shoot proliferation and elongation more effectively. Further, the addition of 0.5μM NAA in the MS medium supplemented with 2.5 μM of BA increased shoot multiplication and elongation frequency from 58 and 17 percent to 65 and 21 percent respectively. The rooting frequency was found to be maximum on 1/2 strength MS medium supplemented with 5.0 μM of indole 3-butyric acid (IBA), which was found to be a superior auxin for inducing roots as compared to the NAA and IAA. With a 75% survival rate, in vitro raised plantlets were effectively acclimatized first in a poly house and later under greenhouse conditions. Molecular analysis was carried out using RAPD markers, with results indicating that the micropropagated plants were genetically identical to the mother plant. The developed micropropagation protocol for E. alba can be used at the commercial level for the mass multiplication of plants.