The invention discloses a method for determining a value of polypeptide content or protein content by using a standard substance. The method comprises the following steps of: separating main components and impurities in a sample to be tested by high performance liquid chromatography; collecting the separated components, hydrolyzing the components into amino acids, and thus obtaining the hydrolyzates of the components; adding isotope labeled stable amino acids into the hydrolyzates of the components, measuring the mass fractions of the amino acids in the components by high performance liquid chromatography-isotope dilution mass spectrometry by taking a national amino acid standard substance as a standard, and calculating the proportionality factors of the amino acids which are obtained by hydrolyzing the main components; hydrolyzing the sample to be tested into the amino acids to obtain the hydrolyzate of the sample to be tested, adding the isotope labeled stable amino acids into the hydrolyzate of the sample to be tested, and measuring the mass fractions of the amino acids in the sample to be tested by the high performance liquid chromatography-isotope dilution mass spectrometry by taking the national amino acid standard substance as the standard; and calculating the mass fractions of polypeptide and protein in the sample to be tested. The method for determining the value of the polypeptide content or the protein content by using the standard substance is high in accuracy, and a value determining result can be finally traced to an international system (SI) unit.