AbstractMedicinal plants, such as Polyalthia longifolia (Indian mast tree), are important therapeutic sources for curing human diseases. In this work P. longifolia leaf extract was characterized by chromatographic and spectral fingerprinting techniques, phytochemical and heavy metal analyses, and microscopy. Light microscopy of a transverse section of the leaf of P. longifolia revealed the presence of various plant cells. Phytochemical screening results revealed the presence of alkaloids, triterpenoids, tannins, saponin, anthraquinones, and glycosides in the extract. The concentrations of heavy metals determined in the extract were well below the permissible limit. Nine peaks observed in the HPLC spectra showed the presence of various compounds in the extract. The GCMS method used for quantification of (3β,4α,5α,9β)-4,14-dimethyl-9,19-cycloergost-24(28)-en-3-yl acetate (i.e., cycloeucalenol acetate) in the extract was rapid, accurate, precise, linear (R2 = 0.8752), and robust. The HPTLC analysis showed ten specific peaks for the methanolic extract of P. longifolia leaf. Twelve major peaks in the range of 4,000 to 500 cm-1 were observed in the FTIR spectra, which represented various specific functional groups in the extract.