Neuropeptides (NPs), a unique and highly important class of signaling molecules across the animal kingdom, have been extensively characterized in the neuronal tissues of various crustaceans. Because many NPs are released into circulating fluid (hemolymph) and travel to distant sites in order to exhibit physiological effects, it is important to measure the secretion of these NPs from living animals. In this study, we report on extensive characterization of NPs released in the crab Cancer borealis by utilizing in vivo microdialysis to sample NPs from the hemolymph. We determined the necessary duration for collection of microdialysis samples, enabling more comprehensive identification of NP content while maintaining the temporal resolution of sampling. Analysis of in vivo microdialysates using a hybrid quadrupole-Orbitrap™ Q-Exactive mass spectrometer revealed that more than 50 neuropeptides from 9 peptide families-including the allatostatin, RFamide, orcokinin, tachykinin-related peptide and RYamide families - were released into the circulatory system. The presence of these peptides both in neuronal tissues as well as in hemolymph indicates their putative hormonal roles, a finding that merits further investigation. Preliminary quantitative measurement of these identified NPs suggested several potential candidates that maybe associated with the circadian rhythm in Cancer borealis.