Background: Hemidesmus indicus is a well known traditional medicinal plant found distributed throughout India. It belongs to Asclepiadaceae family. There are numerous scientific reports on Hemidesmus indicus. The roots of this medicinal plant find extensive used in Ayurvedic medicine. Aim: The present study deals with the fingerprinting of root extract of Hemidesmus indicus Materials and Methods: In this work extractive value, preliminary phytochemical screening, spectral and chromatographic fingerprinting of extract is documented. Results: The polar extracts contain steroids, terpenoids and flavonoids prominently. IR and NMR fingerprints are indicative of the small molecules 2-hydroxy 4-methoxy benzoic acid,2-hydroxy 4-methoxy bezaldehyde, lupeol and amyrin. Conclusion: In view of the need for standardized extracts of Hemidesmus indicus in Ayurvedic formulations, the present study was carried out to fingerprint the root extracts of Hemidesmus indicus. The study will aid herbal practitioners to obtain authentic roots of valuable plant.