Substance abuse, particularly cocaine, has detrimental effects on global public health, as acute intoxication often results in adverse outcomes and current pharmacological treatments exhibit limited effectiveness. The intricate process of cocaine intoxication involves substantial alterations in the brain's neurochemical systems, including fluctuations in neuropeptide levels. To unravel these complexities, we devised an acute intoxication model in rats and undertook a comprehensive brain peptidome analysis using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).