Enicostema axillare (Lam.) A. Raynal, known as the Indian whitehead, is member of Gentiaceae family and has been used medicinally for a very long time both in India and around the world. The objective of the present work comprises collection, identification, macroscopical, microscopical, and phytochemical evaluation and HPTLC fingerprint analysis on Leaves of E. axillare. The surface studies of the leaf are amphistomatic, with anisocytic stomata and trichome complex absent in both surfaces. The T.S of Leaf is slightly flat adaxially and ribbed on abaxially at midvein, presence of one layered epidermis. Mesophyll is heterogenous, differentiated into palisade and spongy tissues, and interspersed with sphaerocrystaliferous idioblasts. The ground tissue of the mid vein consists of palisade 1-2 layered and parenchyma 1-3 layered. The midvein consists of a single oval-shaped vascular bundle, at the center. The microscopic and organoleptic characters of the powder are provided. The phytochemical analysis following hot continuous and successive extraction by Soxhlet apparatus on various extracts indicates the presence of alkaloids, phenolic glycosides, tannins, steroids and terpenoids flavonoids, saponins, phytosterols, fixed oils, gums and mucilage’s, etc., It is discovered that the number of spots, and Rf values of HPTLC are distinct diagnostic features of the plant for identification. The information accumulated through the combination of chemical and botanical screening could potentially be utilized for establishing pharmacopeial standards as well as creating a standard for the identification and authentication of E. axillare.