Abstract: NMRS was applied to the determination of methyl ester of cholic acid. Maleic acid was used as the internal standard. Deuterated dimethyl sulfoxide and deutoxide were used as solvents. The testing temperature taken was 298 K, the pulse width was 8 μs, the relaxation time was 50 s, and the number of scanning was 16. The proton signals at δ 3.79 and δ 0.59 for methyl ester of cholic acid, and at δ 6.28 for maleic acid were selected for quantification. The content of methyl ester of cholic acid was obtained by calculating the average of test results of 2 quantitative peaks. Linearity range of mass ratio of maleic acid and methyl ester of cholic acid was found between 0.208-1.03. The content of methyl ester of cholic acid found was 99.4% with RSD (n=6) of 0.33%.