Aim of this study is to identify and characterize the bioactive principles from the root bark of Calotropis gigantea. It has wide folk medicinal use. For isolation of the compounds, the dried root bark's powder of Calotropis gigantea were subjected to hot extraction and then the crude methanol (MeOH) extract was fractionated with petroleum ether, chloroform and ethyl acetate. Two compounds were isolated and purified from petroleum ether fraction of crude methanol extract and the structures were determined as stigmasterol and beta-sitosterol by analysis of physical, chemical and spectral characteristics (1D NMR and mass spectrometry).