Objective: To develop chemical fingerprint profile of secondary metabolites of therapeutically important plant Elephantopus scaber L. using HPTLC technique. Method: Preliminary phytochemical investigation was performed for the detection of secondary metabolites. HPTLC fingerprint profile of hydro-alcoholic extract of E. scaber was developed for alkaloids, flavonoids and saponins. Toluene: Ethyl acetate: Diethyl amine (7:2:1), Ethyl acetate: Formic acid: Glacial acetic acid: Water (10:0.5:0.5:1.3) and Chloroform: Acetic acid: Methanol: Water (6.4:3.2:1.2:0.8) were the solvent systems developed. Results: The HPTLC chromatogram of hydro-alcoholic extract of E. scaber resolved 12 alkaloids, 25 flavonoids and 26 saponins with different Rf values including some unknown. Conclusion: Chemical profiling of selected plant is suggestive of several known and unknown bioactive molecule having potential therapeutic efficacy. The unknown alkaloids, flavonoids and saponins separated needed to be elucidated further. It can be concluded that the HPTLC fingerprint analysis is useful in exact identification of plant material. Also, it serves as a significant tool in differentiating adulterants and biomarker in herbal drug industry. © 2016, Association of Pharmaceutical Teachers of India. All rights reserved.