A novel strategy combining iTRAQ with 18O stable isotope labeling (iTRAQ plus 18O) was established to identify N-glycosylation site, quantify the glycopeptides and non-glycosylated peptides, and obtain N-glycosylation site ratio on the target glycoprotein. In this approach, all peptides of four biological samples are labeled with four iTRAQ reagents in parallel, followed by PNGase F catalyzed labeling of N-glycosylation sites with H216O and H218O. Two sample groups are labeled with H216O and the other two are labeled with H218O. After the modification of MS precursor ion isolation window, tagged peptides are identified by LC–MS/MS, both glycopeptides and non-glycopeptides are quantified simultaneously using ProteinPilot™ Software. With four samples to be maximally analyzed in parallel, this workflow supports accurate identification and quantification of glycopeptides in a site-specific fashion. Furthermore, N-glycosylation site ratios on serum haptoglobin (Hp) β chain in healthy individuals as well as patients with hepatitis B virus (HBV), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) were quantified to validate the novel ‘iTRAQ plus 18O’ method. Glycosite ratios of VVLHPN#YSQVDIGLIK were observed to change significantly in HCC patients compared with LC and HBV patients. This novel approach supports the screening of the target glycoproteins as biomarkers in clinical application.Highlights► We established a novel strategy combining iTRAQ with 18O stable isotope labeling. ► Glycopeptides and glycosite ratio derived from Hp β chain were quantified. ► VVLHPN#YSQVDIGLIK was observed to change significantly in HCC patients.