<p>N-Glycans represent an important post-translational modification of proteins that can serve as biomarkers of disease, injury, and inflammation. N-Glycosylation of the monoclonal antibody immunoglobulin G (IgG) impacts binding to receptors that initiate an immunological response. Herein, we describe the optimization of a high-throughput method for analyzing IgG glycosylation of multiple murine biofluid samples in a single analysis utilizing matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry. Similar to an enzyme-linked immunosorbent assay (ELISA), our method begins by spotting a capture antibody into a well. However, glycosylation on the capture antibody causes a&#xa0;significant N-glycan background signal that interferes with the signal from IgG-derived glycans in serum samples. To eliminate endogenous capture antibody IgG glycans (i.e., chemical background), the capture antibody was deglycosylated using the enzyme PNGase F, purified using affinity chromatography, and analyzed using ELISAs to confirm there was no loss of binding affinity and selectivity. The performance of the deglycosylated capture antibody was then compared to that of the traditional glycosylated capture antibody using the MALDI IgG N-glycan screening assay. Background subtraction was performed for samples analyzed with both capture antibodies to compare signal intensities before and after background subtraction, which was previously used to correct for the chemical background produced by glycosylated capture antibodies. We show that the use of background subtraction is not necessary with the use of a deglycosylated capture antibody, and that using the deglycosylated capture antibody increases imaging mass spectrometry signal intensity, giving a more sensitive, accurate, and precise analysis of N-glycans present in murine biofluid samples.</p> Graphical abstract <p></p>