Protein glycosylation is a critical post-translational modification that regulates the functions of diverse proteins in eukaryotes. However, analysis of protein glycosylation is hampered by glycoprotein structural diversity both in site occupancy (macroheterogeneity) and glycan structure (microheterogeneity). We have developed variations of SWATH-MS (Sequential Window acquisition of All THeoretical fragment ions Mass Spectrometry) to enable straightforward yet powerful global and targeted analysis of glycoprotein structural diversity. Using Saccharomyces cerevisiae as a model organism, we measured glycan occupancy and structure on a range of glycoproteins in strains with mutations in the N-glycosylation pathway. We observed different degrees of hypoglycosylation in all mutants, including in mutants in regulatory subunits of the oligosaccharyltransferase that catalyses N-glycosylation. The stronger hypoglycosylation phenotypes occurred in strains with defects earlier within the ER lumenal mannosyltransferase steps or glucosyltransferase steps, and only mannosyltransferase mutants showed extensive global changes in glycan structure. Defects in macroheterogeneity therefore did not correlate with defects in microheterogeneity, highlighting the importance of both aspects of glycoprotein structural diversity. We further used these tools to investigate the constraints of co-evolution of a single oligosaccharyltransferase enzyme with diverse glycoprotein substrates in fungi and vertebrates. These results provide a quantitative and qualitative overview of the regulation of oligosaccharyltransferase activity in the presence of truncated glycan donor substrates, and describe outcomes of defects throughout glycan biosynthesis.