Chinese materia medica Tong-Guang-San（Marsdenia tenacissima） is used for treatment of cancer.Developing a bioactive fraction from this plant with optimized components as antitumor agent or chemosensitizer is of great importance.This paper reports results of methodological research for quality control of extract of total glycosides（ETG），a chemosensitive fraction screened out.In our study，ETG at the concentration of 10 μg/mL did not affect the growth of human leukemia cell line K562，human cervical carcinoma cell line HeLa，human hepatoma cell line HepG2 and human oral epidermoid carcinoma cell line KB-3-1（Cell viability＞98%），but when 10 μg/mL ETG was used in combination with paclitaxel，the cytoxicity of paclitaxel against above tumor cells was significantly increased.By employing a Phenomenex Luna C18 column as stationary phase，methanol-0.1% HAc as mobile phase for gradient elution，and 6 ester derivatives of tenacigenin B（1-6） as reference markers，an HPLC method was used for the qualitative analysis of ETG，with well separation of 25 components and identification of the 6 structurally characteristic markers.The summed peak area for 1-6 accounted for 43.39% of the total peak area.The quantitative analysis of reference 1 was also completed，with relative standard deviations（RSD） for peak area，repeatability of measured concentration and stability of sample within 0.16%-0.55%，linear range of 0.86-6.45 μg，and recoveries of 100.4%±1.2%.The method was applied in the determination of total glycosides of Marsdenia tenacissima with reference 1 amount of 0.74%.