In order to improve the quality standard of Marsdenia tenacissima, a quantitative determination method of tenacissoside H was developed using high performance liquid chromatography. The method was carried out on a YMC ODS-H80 (4.6 mm x 250 mm, 4 microm) column eluted with a mixture of acetonitrile and water (50:50) as the mobile phase. The flow rate was 0.8 mL x min(-1) and the column temperature was 35 degrees C. An evaporative light scattering detector (ELSD) was used with the temperature of drift tube set at 60 degrees C and the gas flow rate of nitrogen set at 1.5 mL x min(-1). The calibration curve was linear in the range from 0.5625 to 36.00 microg (r = 0.9998). The average recovery and RSD were 99.41% and 1.8%, respectively. The contents of tenacissoside H in the 11 samples from different habitats varied from 0.201% to 0.862%. The method established in this paper is specific and reliable to control and evaluate the quality of M. tenacissima.