Quantitative analysis of site-specific glycosylation of proteins is a challenging part of glycoproteomic research. Multiple enrichment steps are typically required in the analytical workflows to achieve adequate characterization of the site-specific glycoforms. In spite of recent advances, quantitative workflows need further development. Here, we report a selective and sensitive MS2 followed by further fragmentation in the linear IT-MS analyzer (MS3) multiple reaction monitoring workflow mass spectrometric method for direct analysis of O-glycopeptides in difficult matrix such as serum. Method optimization was performed using two serum glycoproteins, hemopexin (HPX) and sex hormone binding globulin. With the optimized MS3 workflow, we were able to analyze major glycoforms of HPX directly in human serum. Quantification of the minor glycoforms of HPX and glycoforms of sex hormone binding globulin required enrichment of the protein because these analytes were below the sensitivity of the 4000 quadrupole ion trap hybrid mass spectrometer in the complex serum background. In conclusion, we present a quantitative method for site-specific analysis of O-glycosylation with general applicability to mucin-type glycoproteins. Our results document reliable application of the optimized MS3 multiple reaction monitoring workflow to the relative quantification of O-glycosylation microheterogeneity of HPX in human serum. Introduction of isotopically labeled standards would be desirable to achieve absolute quantification of the analytes. The possibility to analyze serum samples directly represents a significant improvement of the quantitative glycopeptide workflows with the potential for use in clinical applications.