N-glycosylation, a crucial post-translational modification (PTM) referring to the attachment of glycan to asparagine residues, has biological importance on facilitating protein folding, controlling intrinsic protein structure, and modulating protein interactions. Despite its importance for fundamental biology and biopharmaceutical industry, the understanding of site-specific N-glycosylation on protein properties and functions is still lacking. Advances in mass spectrometry (MS) technique facilitate global analysis of proteoforms and site-specific measurement of PTMs, providing the possibility to map and quantify site-specific N-glycosylation on proteins. Our study developed a high-throughput strategy to discover specific N-glycosylation sites impacting protein property by thermal profiling of glycoproteome with partially site-specific de-glycosylation. By perturbing the N-glycosylation process, we generated glycoproteoforms with and without specific N-glycosylation sites. Profiling of these partially deficient N-glycosylation sites individually of proteins from native to denatured state under gradient temperatures enabled us to discover functional N-glycosylation sites based on change trend in N-glycosylation occupancy of specific sites derived from the soluble fraction post-thermal treatment. The established method provides an efficient strategy to reveal functional PTMs on a large scale, with potential applications in different systems such as exploring the impact of site-specific glycosylation on protein-protein or protein-ligand interactions, likely benefiting stability enhancement of biologics and biomarkers discovery.