Abstract Purification of glycopeptides prior to the analysis by mass spectrometry (MS) is demanded due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among various purification methods, hydrophilic interaction liquid chromatography (HILIC) has become a popular method in recent years. In this work, we reported a novel magnetic bead-based zwitterionic HILIC (ZIC-HILIC) material which was fabricated by coating a zwitterionic polymer synthesized by spontaneous acid-catalyzed polymerization of 4-vinyl-pyridinium ethanesulfonate monomer on iron oxide magnetic nanoparticles. The resulting magnetic ZIC-HILIC nanoparticles were shown to provide high specificity and high recovery yield (95–100%) for the enrichment of glycopeptides from a standard glycoprotein, fetuin, using a simple magnetic bar. In addition, we proposed a two-step HILIC enrichment strategy using magnetic ZIC-HILIC nanoparticles for a large scale analysis of glycoproteins in complex biological samples. Using this approach, we identified 85 N-glycosylation sites in 53 glycoproteins from urine samples. Two novel glycosylation sites on N 513 of uromodulin and N 470 of lysosomal alpha-glucosidase which have not yet been reported were identified by two-step HILIC approach. Furthermore, all these identified sites were confirmed by studies conducted using PNGase F deglycosylation and 18O enzymatic labeling.