Nicotiana tabacum, plant has been used traditionally 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 current study was therefore carried out to provide requisite pharmacognostic details about the stem 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 Scalariform vessels, Group of fibers, Parenchymatous cells, Spiral xylem vessel, Brownish matter and Phloem fiber. The phytochemical screening revealed the presence of alkaloids, flavonoids, phytosterols, triterpinoids, and tannins. The Rƒ 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.