N-glycosylation is one of the most common and important post-translational modifications, and plays a vital role in the control of many biological processes. The increasing discovery of abnormal alterations of N-linked glycans associated with many diseases leads to greater demands for rapid and efficient N-glycosylation profiling in large-scale clinical samples. In the workflow of global N-glycosylation analysis, enzymatic digestion is the main rate-limiting step, including both protease digestion and PNGase F deglycosylation.