We report here a robust in vitro Reincarnation that is repeatable protocol for the medicinally important plant Enicostemma littorale Blume. Young leaf explants were employed to further develop direct shoot induction, focusing mainly on optimal plant growth regulator combinations and preferred concentrations. All experiments were performed in MS baseline substrate. Numerous meditations on the cytokinins 6-Benzylaminopurine (BAP) and kinetin (KN) were used singly or in concert to cause precocious shoot emergence. The highest number of shoot regeneration was observed over MS medium fortified with 2.22 µM BAP+2.32 µM KN with the highest frequency of shoot induction (92) and maximum number of shoots/explant (20). In contrast, calli production unusual was observed and formed with elevated levels of (BAP (8.88 µM) and KN (4.64 µM), which may be due to a threshold effect which restricted a direct shoot organogenesis. NAA: Induction of roots was tested at different concentrations of auxin naphthaleneacetic acid (NAA). Approximately fifteen roots per shoot were obtained on half-strength MS media containing 1.22 µM NAA (100 % rooting percentage). Downstream research involving the development of the propagation, conservation, and gene improvement of E. littorale at a large scale is expected to be expedited by the rapid and efficient regeneration process of the established protocol. These results are the basis for potential biotechnological applications of this medicinally pure species in the future and emphasize the importance of hormone profile in the context of positive direct organogenesis. published by the Journal of Biodiversity and Environmental Sciences | JBES