The CD44 family are type-1 transmembrane glycoproteins which are important in mediating the response of cells to their microenvironment, including regulation of growth, survival, differentiation, and motility. All these important functions have been reported to be regulated by N-glycosylation; however, little is known about this process. In the CD44 family, the most prolific isoform is CD44 standard type (CD44s). In this work, an integrated strategy combining stable isotope labeling, chemical derivatization, hydrophilic-interaction liquid chromatographic (HILIC) separation, and mass spectrometric (MS) identification was used to perform a comprehensive qualitative and quantitative survey of the N-glycosylation of recombinant CD44s. Specifically, the occupation ratios of the N-glycosites were first determined by MS with (18)O labeling; the results revealed five glycosites with different occupation ratios. Next, N-glycans were profiled by chemical derivatization and exoglycosidase digestion, followed by MALDI-TOF-MS and HILIC-ESI-MS-MS analysis. Interestingly, the quantitative analysis showed that non-sialylated, fucosylated complex-type glycans dominated the N-glycans of CD44s. Furthermore, the site-specific N-glycan distributions profiled by LC-ESI-MS(E) indicated that most glycosites bore complex-type glycans, except for glycosite N100, which was occupied by high-mannose-type N-glycans. This is the first comprehensive report of the N-glycosylation of CD44s. Figure Strategies for characterization of the N-glycosylation status of CD44s.