It has long been appreciated that asparagine (N)-linked glycosylation of proteins is critical for productive glycoprotein folding and high thermodynamic stability. However, it has remained analytically challenging to investigate the precise details of the site-specific roles of N-glycans in determining the stability of glycoproteins. Thermal proteome profiling has emerged as an efficient mass spectrometry-based method for identifying protein-ligand interactions at a proteome scale. Here, we developed a simple sample preparation strategy to enrich yeast glycoproteins from culture supernatant, and show that this subcellular fraction can be used to assay N-glycosylation occupancy and structure. Further, we used this secretome from yeast with defects in N-glycosylation together with a modified thermal proteome profiling approach to efficiently assay the global consequences of site-specific glycosylation defects on glycoprotein thermal stability.