Abstract: The purity of candidate reference materials for creatine and guanidinoacetic acid was investigated using two methods based on different principles: mass balance method and quantitative NMR method. The uncertainties introduced during the development of these reference materials were systematically evaluated. Mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance hydrogen spectroscopy were used for qualitative analysis of the raw materials. Karl Fischer coulometric titration was employed to determine moisture content, ion chromatography for quantification of non-volatile impurities, and headspace gas chromatography for determination of volatile impurity content. High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to analyze impurities with structures similar to the main components. HPLC was employed for homogeneity testing and stability studies. The uncertainties introduced during the development process, including those from weighing, value assignment, homogeneity, and stability, were systematically analyzed and evaluated. The purity values for creatine and guanidinoacetic acid were determined to be 99.30% and 99.30%, respectively, with relative expanded uncertainties of 0.16% and 0.14% (k=2). The developed purity reference materials for creatine and guanidinoacetic acid can provide a traceable basis for the clinical diagnosis of creatine deficiency.