Abstract: N-linked glycosylation, as one of the most common post-translation modification in proteins, is modified on asparagine residue among the N-X-T/S/C(X≠P) motif. N-glycans share a common core structure consisting of two N-acetylglusosamines and three mannoses, and which have three common types of high-mannose, hybrid and complex. N-glycosylation sites have both macro- and micro- heterogeneities and the function of each N-glycoylation is both site- and structure-specific. The precise site- and structure-specific identification and quantification of N-glycosylation is the main road of discovering disease diagnostic and prognostic biomarkers as well as drug targets. With the advancements in high-efficient enrichment materials, liquid chromatography separation technology, cascade mass spectrometry dissociation and detection technologies and bioinformatics database search methods, N-glycoproteomics enables high-throughput site- and structure-specific identification and quantification of N-glycosylation in complex systems. This paper mainly reviewed the basic procedures in the N-glycoproteomics pipeline including sample preparation, high-performance LC-MS/MS analysis and bioinformatics data processing as well as representative applications.