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The separation and determination of lupeol from Hygrophila spinosa were carried out by two simple, precise and accurate HPTLC and RP-HPLC methods. HPTLC method for the determination of lupeol from plant extract and its formulation was developed using a solvent system consisting of toluene : ethyl acetate : methanol (15: 3: 1.5%v/v/v). For detection, lupeol had to be derivatized with Liebermann Burchard reagent at 1050C. The optimum wavelength was fixed as 366nm. In RP-HPLC, the separation was carried out on a C18 column and the mobile phase selected was methanol: acetonitrile (30:70%v/v). The maximum wavelength was found to be 210nm.The method was validated in terms of various parameters. Low relative standard deviation and good % recovery values of both the methods showed that the developed methods were highly precise and accurate and therefore can be used for the standardization and quantification of lupeol in Hygrophila spinosa and its formulation.