Abstract: The aim of this study is to identify and characterized the bioactive compounds from the root bark of the plant. Preliminary phytochemical screening of the root bark extract of Terminalia glaucescens revealed the presence of steroids, terpenoids, saponins, flavonoids, tannins and cardiac glycoside. The plant has wide folk medicinal use in traditional medicine. The air dried root bark was pulverized to powder, subjected to hot extraction (soxhlet) with methanol, and fractionated into n-hexane, ethyl acetate, and n-butanol fractions. Ethyl acetate as bioactive fraction based on sensitivity test was subjected to TLC and column chromatography. The isolated compound was colourless powder, which was further subjected to IR, UV, 13CNMR and 1HNMR for proper characterization and elucidation of the structure. The compound was concluded as β-Sitosterol.