This study introduces the general method of quantitative nuclear magnetic resonance (qNMR) for the calibration of reference standards of macrolide antibiotics. Several qNMR experimental conditions were optimized including delay, which is an important parameter of quantification. Three kinds of macrolide antibiotics were used to validate the accuracy of the qNMR method by comparison with the results obtained by the high performance liquid chromatography (HPLC) method. The purities of five common reference standards of macrolide antibiotics were measured by the 1 H qNMR method and the mass balance method, respectively. The analysis results of the two methods were compared. The qNMR is quick and simple to use. In a new medicine research and development process, qNMR provides a new and reliable method for purity analysis of the reference standard.