A bioactive compound of Phyllanthus amarus Schum et Thonn. phyllanthin in name, was isolated and pruified. The confirmation of its structure was mainly done by UV-Vis, IR, MS, 1HNMR, 13CNMR. In HPLC analysis, calibration curves established on six replicate experiments with phyllanthin demontrated good linear regression over a range of 4 g/ml - 20 g/ml with R2 = 1. The precision RSD was less than 2 percent. The accuracy of phyllanthin ranged from 99.03- 100.03 percent (averaged at 99.47 percent). It was traceable to the corresponding certified reference material of Chromadex in terms of content to serve as a botanical reference standard for quality control and research of the plant materials.