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Abstract Glycosylated neuropeptides were recently discovered in crustaceans, a model organism with a well‐characterized neuroendocrine system. Several workflows exist to characterize enzymatically digested peptides; however, the unique properties of endogenous neuropeptides require methods to be re‐evaluated. We investigate the use of hydrophilic interaction liquid chromatography (HILIC) enrichment and different fragmentation methods to further probe the expression of glycosylated neuropeptides inCallinectes sapidus. During the evaluation of HILIC, we observed the necessity of a less aqueous solvent for endogenous peptide samples. This modification enabled the number of detected neuropeptide glycoforms to increase almost two‐fold, from 18 to 36. Product ion‐triggered electron‐transfer/higher‐energy collision dissociation enabled the site‐specific detection of 55 intact N‐ and O‐linked glycoforms, while the faster stepped collision energy higher‐energy collisional dissociation resulted in detection of 25. Additionally, applying this workflow to five neuronal tissues enabled the characterization of 36 more glycoforms of known neuropeptides and 11 more glycoforms of nine putative novel neuropeptides. Overall, the database of glycosylated neuropeptides in crustaceans was largely expanded from 18 to 136 glycoforms of 40 neuropeptides from 10 neuropeptide families. Both macro‐ and micro‐heterogeneity were observed, demonstrating the chemical diversity of this simple invertebrate, establishing a framework to use crustacean to probe modulatory effects of glycosylation on neuropeptides.