Proteins and sugars are important components of food. During food processing, storage and transportation, the amino group of protein molecules is easily combined with the carbonyl group of reducing sugar through covalent bonds, and the glycation reaction occurs. Maillard reaction-based glycation includes three stages: the early stage, the middle stage and the advanced stage. The glycation reaction modifies the main or side chains of proteins, changes the structure, static charge and hydrophobicity of proteins, and affects the chemical activities of the main and side chain groups of proteins, thus changing the physicochemical properties of proteins, including improving the emulsifying, foaming, gel and other functional properties, enhancing the antioxidant and other nutritional properties, while reducing the sensitization and enhancing the antibacterial and storage properties. It is very common and important in food processing, storage and transportation. The changes in protein structure during the reaction are the important causes of the alteration of nutritional and functional properties. In recent years, chemical analysis (free amino content and surface hydrophobicity determination, etc.), spectrometry (ultraviolet spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, etc.), chromatography (circular dichroism, size-exclusion high-performance liquid chromatography, etc.), mass spectrometry (matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), liquid chromatography-electrospray tandem mass spectrometry (LC-MS2), etc.) and other techniques have been explored to analyze the structural changing rules of protein during glycation reaction, playing the important roles in the mechanism study of glycation reaction as well as regulation of glycation degree and nutritional and functional properties of glycation products. In particular, the emergence and mature application of orbital ion trap mass spectrometry (LC-Orbitrap-MS2), Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS2), hydrogen-deuterium exchange Fourier transform ion cyclotron resonance mass spectrometry (HDX-FTICR-MS2), and other technologies have made the research on glycation sites more in-depth. These technologies make it possible to reveal the mechanism of protein glycation reaction by quantifying the number of bonded sugar molecules and the degree of glycation substitution at each reaction site. This study reviewed characterization and detection methods for the protein glycation degree, primary, secondary and tertiary protein structures and functional group structure. This study aims to provide a reference for the deep research of protein glycation reaction and its application in food processing.