Neuropeptides are important signaling molecules that regulate many essential physiological processes. Microdialysis offers a way to sample neuropeptides in vivo. When combined with liquid chromatography-mass spectrometry detection, many known and unknown neuropeptides can be identified from a live organism. This chapter describes sample preparation techniques and general strategies for the mass spectral analysis of neuropeptides collected via microdialysis sampling. Methods for the in vitro microdialysis of a neuropeptide standard as well as the in vivo microdialysis sampling of neuropeptides from a live crab are described.