Abstract: N-linked glycosylation is a common post-translational modification on proteins, which exhibits the same macro-heterogeneity of modification site as other small molecule modifications (such as methylation, acetylation, phosphorylation), i.e., the amino acid sequence of a protein has multiple putative modification sites. However, compared to small molecule modifications with single structures, N-glycosylation modification have tens of thousands of structures from multiple structural dimensions such as different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformation.This results in additional micro-heterogeneity of modification site of N-glycosylation, i.e., the same N-glycosylation site can be modified with different glycans with a certain stoichiometric ratio.N-glycosylation modification regulates the structure and function of N-glycoproteins in a site- and structure-specific manner, and differential expression of N-glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis.This article mainly introduces the latest development of mass spectrometry-based site- and structure-specific quantitative N-glycoproteomics and its applications in biomedical fields.