Background: Fruits of Benincasa hispida (BH) is regarded as Valliphala due to its vast plethora of medicinal properties, Carissa congesta (CC) is an imperative local plant particularly in rural communities and Polyalthia longifolia (PL) is an ornamentally significant traditionally relevance plant in India system. β-sitosterol, an active constituent identified from enormous plants has been reported to possess excellent amount of pharmacotherapeutic potential by number of researchers. Objective: In the recent studies, the research team focuses on determining the percentage of the β-sitosterol present in the BH seeds, CC roots petroleum ether extracts as well as PL leaves ethanolic extract by chromatographic technique in harmony with High Performance Thin Layer Chromatography. Materials and Methods: Respective parts of BH, CC and PL plants were shade-dried and extracted by appropriate extraction methods followed by identification of β-sitosterol from the extracts by High Performance Thin Layer Chromatography after preliminary phytochemical screening extracts for the constituents. Results: The amount of β-sitosterol present in the BH seeds, CC roots and PL leaves extracts was found to be 23.00, 5.94 and 1.81 % w/w respectively. Research studies elucidated a peak that coincided with standard peak of β-sitosterol suggesting the presence of constituent in the extracts. Conclusion: Thus, extracts contains important constituent of β-sitosterol in BH, CC and PL.