Updated project metadata. Glycosylation, a common and heterogeneous post-translational modification plays a key role in altering biomolecule function and other properties. Prior to monitoring regulatory roles, basal glyconeuropeptide expression must be determined. Characterization is partially limited by the lower abundance of glycosylated neuropeptides in vivo compared to non-modified neuropeptides and thus requires effective enrichment and characterization strategies. A hydrophilic interaction liquid chromatography enrichment strategy is modified, and fragmentation schemes are evaluated to establish a workflow for future glyconeuropeptide studies to improve the understanding of neuropeptide glycosylation, as well as its distribution across the neuroendocrine system and several neuropeptide families. Product ion-triggered electron-transfer/higher-energy collision dissociation and stepped collision energy higher-energy collisional dissociation are evaluated.