Objective: To investigate pharmacognostical and physico-chemical standardization of Euphorbia neriifolia leaves. Materials and Methods: Fresh and dried leaves with powder samples of E. neriifolia were examined macroscopically and microscopically. As per Ayurvedic Pharmacopeia of India and World Health Organization guidelines on quality control methods for medicinal plants materials suggested parameters were determined for standardization of E. neriifolia leaves. Physico-chemical, primary phytochemical, fluorescence and quantitative screenings along with primary HPTLC fingerprinting assessment were performed. Results: Macroscopic examination demonstrated that fresh leaf of E. neriifolia has dark green in colour, herbaceous odour with characteristic taste. Dried leafs are grey brownish in colour, characteristic odour with broken crumpled and papery fracture. Microscopy of leaf showed the single layered thick rectangular or tubular adaxial epidermal cells. Mesophyll tissue was differentiated into two or three layered adaxial zones of radially elongated palisade cells and wider abaxial spongy mesophyll cells revealed the differentiated dorsiventral lamina. Mid-rib composed of epidermis, collenchymas and spongy parenchyma cells. Physico-chemical parameters like, foreign matter was found to be 0.46%. Total ash, acid insoluble ash and water soluble ash was found 6.33%, 1.23% and 6% respectively. Loss on drying was found to be 4.69%. Swelling and foaming index was found 11.7 ml and 333 ml respectively. Quantitative screening suggested that the leaf powder has indicated alkaloid and saponin estimation as 0.26% and 3.67% respectively. The HPTLC fingerprinting of EN6 extract fraction was showed the Rf values at 254 nm with their respective UV-visible spectrum wavelengths scanned in between 200-400 nm. They are 0.01 (265 nm), 0.05 (369 nm), 0.09 (263 nm, 264 nm), 0.18 (400 nm), 0.20 (279 nm), 0.31 (400 nm), 0.44 (378 nm), 0.45 (382 nm), 0.54 (377 nm), 0.55 (383 nm), 0.62 (400 nm), etc. at different concentrations of sample application. The HPTLC plate was also scanned at 366 nm and 540 nm. Conclusion: The present investigation is an additional standardization research in support with previous reports and will be helpful for qualitative and quantitative standardization of herbal formulations containing E. neriifolia. Further investigations are going on this extract fraction in reference to identification, quantification and validation of HPTLC methods using various standard marker compounds along with exploration of its pharmacological activities.