A high performance liquid chromatography method was established for quantitative determination of disodium 5′-inosinate and disodium 5′-guanylate (IMP and GMP). Using tetrabutylammonium phosphate monobasic and potassim dihydrogen phosphate mixed with a certain proportion of acetonitrile as the mobile phase, the standard samples were analyzed on Waters RP 18 column under the optimal conditions. The qualitative and quantitative reproducibility of RSD were below 2%. On the IMP standard curve, the linear range was 60~600μg/mL, the correlation coefficient R being 1.000. On the GMP standard curve, the linear range was 50~480μg/mL,the correlation coefficient R being 0.9999. The method was used to determine IMP and GMP content in a certain brand of chicken extract, which was 0.54% and 0.60%, respectively, the precision being 0.73% for GMP, 1.17% for IMP.