Abstract: The purity of the standard candidates of creatine and guanidinoacetic acid was studied by two methods with different principles, namely, mass balance method and quantitative NMR method, and the uncertainties introduced during the development of the standards were systematically evaluated. Mass spectrometry, infrared spectroscopy and nuclear magnetic resonance hydrogen spectrometry were used to characterize the raw materials of the standards; Karl Fischer coulometric titration was used to determine the moisture content, ion chromatography was used to quantify the non-volatile impurities, and headspace gas chromatography was used to determine the content of volatile impurities; high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to analyze the structurally similar impurities to the main components; and HPLC was used for uniformity test and homogeneity test and tandem mass spectrometry. High performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to analyze the impurities similar to the structure of the main components. At the same time, the uncertainties introduced by weighing, calibration, homogeneity and stability during the development of the standards were systematically analyzed and evaluated, and the results of the purity calibration of creatine and guanidinium acetic acid were 99.30% and 99.3%, respectively, with the relative extended uncertainties of 0.16% and 0.14%. The developed purity standards of creatine and guanidinoacetic acid can provide a traceable basis for the clinical diagnosis of creatine deficiency.