Macrolides are multicomponent drugs whose impurity control is always a challenge demanding analysis method with good sensitivity and selectivity. Three separate, sensitive, accurate liquid chromatography tandem mass spectrometry methods (LC–MS) were developed for the measurement of three 16-membered ring macrolides (josamycin, josamycin propionate and midecamycin acetate) and related substances in commercial samples. The characteristics of impurities in macrolides were summarized as useful guidance for the impurity analysis of this class of drugs. For each drug, a large number of unknown components have been detected with the high-sensitive MS detector and possible structures of the majority of them were postulated based on the summarized fragmentation rules of 16-membered ring macrolides. A QSRR model was constructed by multilinear regression to predict the retention times of identified impurities which were not detected by the LC–MS methods, without obtaining their reference standards. Satisfactory performance was obtained during leave-one-out cross-validation with a predictive ability (Q2) of 0.95. The generalisation ability of the model was further confirmed by an average error of 2.3% in external prediction. The best QSRR model, based on eight molecular descriptors, exhibited a promising predictive performance and robustness.