Abstract: The study aims to establish a quantitative nuclear magnetic resonance (QNMR) method for the determination of the absolute content of bosentan. Proton nuclear magnetic resonance spectroscopy ¹H NMR spectra were obtained in CDCl₃ with the internal standard dimethyl terephthalate and zg30 pulse sequence by using a Bruker AVANCE Ⅱ 400 spectrometer. The content of bosentan is determined with QNMR in comparison with the result obtained by mass balance method. The result is 96.25% by QNMR and 96.54% by mass balance method. A rapid and accurate QNMR method has been established for the quantitative determination of the absolute content of bosentan. The study provides a new way for the quality control and calibration of a new reference standard material, it could be the complementary with the mass balance method for the assay of standard reference.