Abstract Copper is an essential nutrient with critical roles in protein structure and enzyme activity but becomes toxic at elevated levels due to its ability to participate in redox reactions. Copper toxicity is a growing concern as environmental copper concentrations increase due to agricultural and industrial runoff and poorly managed wastewater. The dysregulation of copper homeostasis has been linked to many diseases including anemia, liver damage, and neurodegenerative diseases, but the exact mechanisms are not fully understood. The biochemical response to copper toxicity has been shown to involve neuropeptides, but the full suite of relevant neuropeptides and their expression changes are difficult to characterize due to the complexity of the nervous system. Crustaceans provide a highly relevant model organism with a simple, well-characterized nervous system, facilitating meaningful analysis of environmental stress. Neuropeptides remain challenging to study, however, due to their structural diversity and low in vivo concentrations. As a result, mass spectrometry (MS) has become the preferred method of analyzing neuropeptides. MS is highly sensitive and selective, capable of providing both structural and quantitative information, and requires no prior knowledge of the analytes in the sample. This enables the analyses of not only neuropeptides, but also the chelating molecules responsible for maintaining homeostasis, such as glutathione and metallothionein proteins. To thoroughly probe the biochemical and physiological response to copper toxicity, I propose to: 1) develop and optimize high-throughput, multiplexed mass spectrometry techniques to quantify neuropeptides, 2) globally profile the neuropeptides involved in the stress response to both chronic and acute copper toxicity, and 3) analyze the expression changes of copper chelating molecules in the gills, hemolymph, and hepatopancreas using top-down MS methods. The proposed research will provide a deeper understanding of the signaling molecules and biochemical defense mechanisms involved in responding to excess copper, thereby providing a better understanding of how environmental stressors (such as heavy metals) can have profound impacts on health. Moreover, understanding the stress response can provide unique insights into the many diseases linked to copper toxicity and dysregulation of copper homeostasis.