Chromatographic techniques can be used to document phytochemical fingerprints and quantitate chemical markers to identify morphological and geographical variations in the herbal raw material. In this context, phytochemical profile of Asteracantha longifolia Nees. (syn. Hygrophila spinosa T. Anders.; Hygrophila auriculata [K. Schum.] Heine), an important medicinal herb, was developed using HPTLC technique and was successfully used for evaluating regional and morphological variations. The HPTLC fingerprints were also used for the quantitation of two bioactive markers β-sitosterol and Lupeol in the plant powder. These phytochemical markers were also evaluated from different parts of the plant and from the whole plant collected from different geographical regions. Maximum content of Lupeol was found to be in roots (0.25%), while maximum content of β-sitosterol was found to be in the leaves (0.069%) of Asteracantha longifolia Nees.