N-glycosylation, a crucial post-translational modification (PTM) referring to the attachment of glycans to asparagine residues, is important in facilitating protein folding, controlling intrinsic protein structure, and modulating protein interactions. Despite its importance for fundamental biology and the biopharmaceutical industry, our understanding of the impact of site-specific N-glycosylation on protein stability is still lacking. Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) facilitates global analysis of proteoforms and site-specific measurement of PTMs, providing the possibility to map and quantify site-specific N-glycosylation on proteins at large scale and high sensitivity. Our study developed a high-throughput strategy to discover specific N-glycosylation sites that impact protein stability using thermal glycoproteome profiling of together with partial site-specific de-glycosylation, and allowed us to discover functional N-glycosylation sites. The established method provides an efficient strategy to reveal functional PTMs affecting protein stability at a large scale, with potential applications in different systems including exploring the impact of site-specific glycosylation on protein-protein or protein-ligand interactions, which will benefit quality enhancement of biologics and discovery of biomarkers.