The amide hydrogen in the protein backbone exchanges with protons in the solvent, and this exchange rate reflects the protein’s three-dimensional and dynamic structure. In the H-D exchange method, the protein is exposed to heavy water to cause an exchange reaction of amide hydrogen to deuterium (H-D exchange), and by observing the state of H-D exchange at each site in the protein by mass spectrometry or nuclear magnetic resonance method, information such as the dynamic structure and interaction site of the protein in solution can be obtained. In particular, hydrogen deuterium exchange mass spectrometry (HDX-MS), which combines the H-D exchange method and mass spectrometry, can obtain structural information of proteins without an upper limit of molecular weight with high sensitivity and is used in many fields such as structural biology. In HDX-MS, the analysis is performed in steps of stopping the exchange reaction, digestion, LC-MS, and data analysis after diluting the target protein with heavy water (see Fig. 15.1). The steps after the reaction stop are performed at low temperature to suppress the back exchange from labeled deuterium to hydrogen. Furthermore, to quench the back exchange, digestion needs to be performed at around pH 2.5 where the exchange reaction is the slowest, so pepsin is used as the protein digestive enzyme. The analysis is completed in about 2 weeks, making it a high-throughput analysis method compared to other structural analysis methods.