We have demonstrated that simultaneously deglycosylating and digesting protein samples on an S-Trap improves the identification of potential N-glycosylated peptides. Importantly, by coincubating PNGase F and trypsin on S-Traps, we can perform this sample preparation in the same amount of time as a standard protein digestion. Identifying peptides that appeared in only deglycosylated samples, had a deamidation modification, and contained an N-glycan motif was a fast and effective way to generate a hypothesized list of glycopeptides. Future experiments could use this method to discover potential biomarkers in disease such as cancers, where aberrant glycosylation is known to play a role. We anticipate using this technique in our future work to study glycosylation in samples from ovarian cancer patients to determine whether differential glycosylation is present throughout the proteome. Other proteins in a complex proteome with N-glycosylation such as monoclonal antibodies and the Rh blood group proteins could be studied using this method as well. Additionally, with the use of suitable enzymes, this technique could be extended to the more difficult, but important, question of O-linked glycosylation on proteins of interest.