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Starvation is a complex physiological state that induces changes in protein expression to ensure survival. The insect midgut is sensitive to changes in dietary content as it is at the forefront of communicating information about incoming nutrients to the body via hormones. Therefore, a DIA proteomics approach was used to examine starvation physiology and, specifically, the role of midgut neuropeptide hormones in a representative lepidopteran, Manduca sexta. Proteomes were generated from midguts of M. sexta fourth-instar caterpillars, starved for 24 h and 48 h, and compared to fed controls. A total of 3047 proteins were identified, and 854 of these were significantly different in abundance. KEGG analysis revealed that metabolism pathways were less abundant in starved caterpillars, but oxidative phosphorylation proteins were more abundant. In addition, six neuropeptides or related signaling cascade proteins were detected. Particularly, neuropeptide F1 (NPF1) was significantly higher in abundance in starved larvae. A change in juvenile hormone-degrading enzymes was also detected during starvation. Overall, our results provide an exploration of the midgut response to starvation in M. sexta and validate DIA proteomics as a useful tool for quantifying insect midgut neuropeptide hormones.