Congenital disorders of glycosylation (CDG) are a family of N-linked glycosylation defects associated with severe clinical manifestations. In CDG type-I, deficiency of lipid-linked oligosaccharide assembly lead to the under-occupancy of N-glycosylation sites on glycoproteins. Although the level of residual glycosylation activity is known to correlate with the clinical phenotype linked to individual CDG mutations, it is not known whether the degree of N-glycosylation site occupancy by itself correlates with the severity of the disease. To quantify the extent of underglycosylation in healthy control and in CDG samples, we developed a quantitative method of N-glycosylation site occupancy based on multiple reaction monitoring LC- MS/MS. Using isotopically labeled standard peptides, we directly quantified the level of N-glycosylation site occupancy on selected serum proteins. In healthy control samples, we determined 98-100% occupancy for all N-glycosylation sites of transferrin and 1-antitrypsin. In CDG type-I samples, we observed a reduction in N-glycosylation site occupancy, which correlated with the severity of the disease. In addition, we noticed a selective underglycosylation of N-glycosylation sites, indicating preferential glycosylation of acceptor sequons of a given glycoprotein. In transferrin, a preferred occupancy for the first N-glycosylation site was observed and a decreasing preference for the first, third and second N-glycosylation site was observed in 1-antitrypsin. This multiple reaction monitoring LC-MS/MS method can be extended to multiple glycoproteins, thereby enabling a glyco-proteomic survey of N-glycosylation site occupancies in biological samples.