Here, we show that PNGase F can be directly coupled with proteolytic digestion using suspension trapping (S-Traps), a powerful tool for proteomics sample processing. In S-Trap sample preparation, an SDS-solubilized protein solution is reduced and alkylated before acidification and addition of methanol to create a protein suspension. The suspension is then centrifuged through the S-Trap spin column and proteins are retained on a quartz filter, which can then be washed free of contaminants, and a protease of choice can be added to begin digestion. S-Traps are most commonly used with trypsin, but can also be used with other enzymes such as Lys-C 31 and GluC. S-Trap sample preparation has been used in a variety of applications including in protein extraction from mouse microglia cells and proteome comparison of the epiretinal membrane and inner limiting membrane from human retinal cells. The use of S-Traps for simultaneous enzymatic deglycosylation and proteolytic digestion has not yet been reported. We demonstrate that PNGase F deglycosylation and tryptic digestion can occur simultaneously on S-Traps, enabling an optimized and efficient sample preparation workflow that takes the same amount of time as standard proteolytic digestion. We focus on Nglycosylation because of the ease of large-scale complex protein analysis due to the predictable, straightforward sites of N-glycosylation and availability of universal enzymes to specifically remove N-glycans from the peptide backbone. By comparing deglycosylated samples with non-deglycosylated (control) samples, we can quickly hypothesize which peptides may contain N-glycans based on their amino acid sequences. These potential glycopeptides can then be followed up with additional analysis.