Abstract: Glycosylation is one of the most important post-translational modifications (PTM), mediating physiological processes like cell adhesion, cell proliferation, cell signaling, and immune responses. The special functionality of glycosylation for cancer progression and metastasis were reported in recent researches. Analytical methods based on mass spectrometry (MS), especially liquid chromatography mass spectrometry (LC-MS), are the routine practices for protein glycosylation research. However, due to the high complexity and multiformity of glycan structures, as well as the existence of numerous isomers, in-depth analysis of glycosylation ends up as a major challenge for conventional LC-MS-based method. Ion mobility separation is a promising approach to fill the gap as it provides an additional separation dimension for mass spectrometry analysis based on structures and charges of the gas-phase ions. As a technology with an entirely different separation mechanism from that of liquid chromatography, ion mobility spectrometry (IM) has several properties superior to other separation facilities. First of all, unlike liquid chromatography or capillary electrophoresis which demand minutes of separation time, IM only requires a few milliseconds to finish the separation, and it is well compatible with fast mass spectrometry analysis. Besides, as a separation technique for gas phase ions, IM can be modularized and readily integrated into full-fledged LC-MS system. Most importantly, IM is proved to have great potential in glycan isomer discriminations. Given the aforementioned merits, ion mobility mass spectrometry (IM-MS) is gaining attention from glycomics community. This review summarized the basic principles of IM-MS and its applications in protein glycosylation research for the past five years. The means of enhancing isomeric separation performance of glycans were comprehensively discussed, including derivatization, forming metal and other noncovalent adducts, and utilizing high-resolution ion mobility spectrometry implementation. The various approaches of leveraging collision cross section (CCS) values to assist glycan identification and isomer discrimination were also summarized. Furthermore, as two monosaccharides with great biological and pathological significance, the IM-MS applications on fucoses and sialic acids were given special attention. As for glycopeptides, innovative spectra matching algorithms, novel ion activation techniques to facilitate glycan and peptide elucidations, and brand new quantification strategies for isomers were included, along with various glycoproteomic applications of IM-MS on biological samples. Finally, in terms of IM-MS applications in glycoprotein research, investigations of conformational properties, 3D structures, stabilities, and protein-protein interactions were covered. It was concluded that IM-MS is a promising method for protein glycosylation research with great potential.