Macrolide antibiotics are broad-spectrum, with activity against a range of Gram-positive, Gram- negative organisms and some anaerobes.  The components of macrolide antibiotics are generally complicated.  Therefore, it is very important to establish impurity profiles of these antibiotics to ensure their safety and process control.  Compared with classical methods, the liquid chromatography-mass spectrometry method is particularly advantageous to characterize minor components at trace levels in terms of sensitivity, efficiency and selectivity, thus more and more widely used in establishments of impurity profiles.  In this study, the general approaches to characterize minor components in complex pharmaceutical matrix, fragmentation pathways of 14- and 16- membered macrolide antibiotics and the establishment of the impurity profile of acetylspiramycin were given to provide valuable enlightenments to establish the impurity profiles of pharmaceutical products.