One of the most critical post-translational modifications in proteins is N-glycosylation, influencing the folding and function of ~1/3rd of the eukaryotic proteome. N-glycosylation occurs when a preassembled glycan is transferred to a protein acceptor at specific protein sites by the oligosaccharyltransferase (OTase). Alterations in glycan structure and occupancy at specific sites have significant clinical and industrial impact, leading to a growing number of diseases (Congenital Disorders of Glycosylation, cancer, etc) and to changes in secretion, half-life, and activity of proteins with biotechnological relevance. A better understanding of the causes and consequences of N-glycosylation changes will lead to more effective therapeutics, diagnostic strategies, and industrial bioprocesses. Thus, to more accurately study the mechanism of N-glycosylation and its physiological impact, we designed several biochemical and quantitative SWATH-mass spectrometry workflows. These workflows allow us to measure site-specific and global changes in glycan occupancy and structure. We applied these tools to study the function of the OTase and to look for glycobiomarkers in clinical samples. We used yeast and mammalian cells with normal or altered glycan biosynthetic processes, and serum samples of patients with potential glycosylation disorders. Our glycoproteomic protocols allow for rapid relative quantitative and qualitative glycoproteomics of a variety of samples. Our results provide insights into the regulation of site-specific glycosylation and the function of the OTase. Our methods and results have important implications in the understanding of the fundamentals of the glycosylation process and on the industrial and medical applications of glyco-biotechnology.