The rapid and effective digestion of proteins is important when performing protein characterization by hydrogen deuterium exchange mass spectrometry (HDX-MS). Several proteins are difficult to digest under low pressure conditions, resulting in low sequence coverage and the need for high concentrations of denaturing and reducing agents. These agents are known to interfere with downstream mass spectrometry analysis. In this study, an enhanced pressure sustainable BEH Enzymate™ digestion column with immobilized pepsin has been applied to the Waters ACQUITY UPLC™ M-Class system with automated HDX technology fitted with a back pressure regulator. High pressure (12 000psi) online peptic digestion of a heavily glycosylated protein with multiple disulphide bonds, led to increased digestion efficiency when compared to low pressure online digestion. High pressure digestion yielded an increased number of overlapping peptides, which resulted in greater protein sequence coverage and higher spatial resolution when compared to lower pressure digestion. High pressure digestion also enabled more effective destabilising effect of chaotropic agents and permitted their use in lesser concentrations that are more compatible with downstream mass spectrometry steps.