Technique advances in proteomics including sample separation, mass spectrometry and searching algorithm have empowered in-depth discovery of protein post-translational modifications from biological samples. Instead, there is still a relative delay in the systematic researches for functional significance of these modifications. Herein, we propose Refined-TPP, a novel methodology for thermal proteomics, to systematically examine the effects of site-specific modification on the thermal stability of native proteins. This globally biophysical assay bridges the gap between the structural modifications and their functional impacts on proteins, and provides here a robust platform for the first time to effectively interrogate the functional implications for N-glycosylation. Finally, we elucidated the pivotal roles of explored N-glycosylation in protein structure and function, including protein stability, subcellular localization and enzyme activity. This novel strategy is readily applicable in a high-throughput and unbiased manner to further investigations for diverse objects, including drug-target screening, protein interactions and other functional PTMs exploration.