Introduction: Gymnema sylvestre R. Br. (Family: Asclepiadaceae) commonly called as Madhunashini (Sugar Killer) in Sanskrit is a high valued medicinal plant due to its principal phytoconstituent Gymnemic acid mainly found in its leaves. Methods: Gymnema sylvestre R. Br. leaf, its powder and leaf extracts were studied using various standardization parameters like macroscopical, microscopical, physicochemical, phytochemical and fluorescence analysis. Further, HPTLC fingerprint profile was developed for methanolic leaf extract. Results: Macroscopically, the leaves were simple, petiolate, broadly ovate in shape, acute apex and rounded base with reticulate venation. Microscopically, the leaf showed the presence of fan shaped vascular bundle, epidermal cells covered trichomes, xylem lying towards upper and phloem towards lower epidermis. Powder microscopy of leaves revealed the presence of vessels, calcium oxalate crystals, fibers, etc. The study also included quantitative leaf microscopy, histochemical and fluorescence analysis. Physicochemical analysis revealed that the water-soluble extractive value of leaf was three times higher than alcohol soluble extractive value. Preliminary phytochemical screening showed the presence of various primary and secondary metabolites viz. Aleurone grains, Alkaloids, Flavonoids, etc. A developed HPTLC fingerprint profile of methanolic leaf extract is unique for Gymnema sylvestre R. Br. and can be used as an identifying marker. Conclusion: The data generated from the present pharmacognostical studies are significant for establishing correct identification, standardization and authentication of genuine plant material.