Abstract: Glycosylation plays important role in cell-cell interaction, signal transduction and cancer immunology, which is currently a hotspot in biomarkers screening for many major human diseases. Though commonly used in biomarker studies, plasma proteome/glycoproteome is under strict homeostatic control, which makes it difficult for biomarker discovery. As the partial filtrate of blood, urine serves not only as an ideal source of biomarker for diseases diagnosis of the urogenital system, but also as a potential source of information on physiological conditions. Therefore, in-depth analysis of human urine glycoproteome is of great significance. However, due to the low abundance of N-glycoprotein in human urine, limited modification stoichiometry and microheterogeneity of N-glycosylation, it is a highly-challenging task for N-glycoprotein/glycopeptide identification by mass spectrometry. Therefore, the development of efficient and selective enrichment method is a prerequisite for large-scale identification of urinary protein N-glycosylation. In this study, we synthesized a novel zwitterionic stationary phase with high hydrophilicity based on the “thiol-ene” click reaction between 3-(methacryloylamino) propyl dimethyl (3-thiopropyl) ammonium hydroxide (SPP) and mercapto silica. SPP-silica exhibited great potential in the enrichment of N-glycopeptides, while keeping the merit of a facile and time-saving synthesis protocol. To evaluate the practicability of SPP-silica in the selective enrichment of N-glycopeptides, a tryptic digest of standard glycoprotein (human IgG) was employed as the testing sample. 36 glycopeptides were captured and identified. SPP-silica showed remarkable selectivity for N-linked glycopeptides even in the presence of 10-fold excess tryptic digests of non-glycoprotein (BSA). SPP-silica was successfully applied to the HILIC enrichment and mass spectrometry identification of urinary protein N-glycopeptide from healthy adults. The same sample was enriched by three batches of SPP-silica and analyzed by mass spectrometry. 959, 837 and 877 of N-glycosylation sites were identified, respectively with >70% selectively. A total of 1 065 N-glycosylation sites in 633 urinary glycoproteins was identified, which is 12.2% larger than that reported in the literature. 81.2% and 73.9% of the obtained N-glycoproteins and sites were identified in at least two experiments. The above results demonstrate the application potential of this hydrophilic material in urinary glycoproteomics research and biomarker screening.