Nicotiana tabacum, a traditional medicinal plant which is valued for its benefits as sedative, laxative, tonic , emetic, carminative, antispasmodic and vermifuge, and in management of skin diseases, local infections, bronchitis, asthma and inflammation. The leaves has been employed in curing old ulcers and painful tumors and for the extraction of the active principle i.e. nicotine which, usually in the form of sulphate, is widely used as an insecticide and in the production of synthetic nicotinic acid and nicotinamide. The current study was therefore carried out to provide requisite pharmacognostic details about the leaves of Nicotiana tabacum. Pharmacognostic evaluation included examination of morphological and microscopical characters; physicochemical properties, phytochemical analysis, and HPTLC fingerprint. The powder microscopy showed the presence of glandular trichome (single celled head and multicellular stalk), covering trichome (multicellular unisereate covering trichome), paracytic stomata, acicular and prismatic calcium oxalate crystals and spiral vessel with bordered thick wings. The phytochemical screening revealed the presence of alkaloids, flavonoids, phytosterols, triterpinoids, tannins and carbohydrates. The Rf values detected at 400 nm by qualitative densitometric HPTLC fingerprint, can be used as identifying marker for petroleum ether extract. The present study will provide the information with respect to identification and authentication of crude drug.