N-Glycosylation, being one of the most common and complex protein post-translational modifications (PTMs), is known to have microheterogeneity with the presence of different N-glycan structures at a single specific glycosite. These different structures may have exactly the same monosaccharide composition but totally different differential expressions and pathological relevance. Mass spectrometry-based N-glycoproteomics has so far been successful in large-scale characterization of these N-glycans at the composition level, and structure-level identification and quantitation is urgently needed. Here we report our development of the intact N-glycopeptide search engine GPSeeker and the GPSeeker-centered quantitative structural N-glycoproteomics pipeline. In analysis of differential N-glycosylaiton in MCF-7 cancer stem cells (relative to MCF-7 cells) with RPLC-MS/MS (HCD) analysis, 2,558 intact N-glycopeptides were identified corresponding to 727 N-glycosites, 724 unique peptides, 136 putative N-glycan linkages from 53 monosaccharide compositions and 640 intact N-glycoproteins; with isotopic diethyl labeling, 144 differentially expressed intact N-glycopeptides with >=1.5-fold change (p<0.05) were quantified. For the 20 known CD series CSC markers, 13 (CD13, CD14, CD20, CD4, CD90, CD63, CD49F, CD151, CD97, CD44, CD49b, CD109 and CD133) were either quantified, identified, or observed as GPSMs.