Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS); its cause is unknown. To understand the pathogenesis of MS, researchers often use the experimental autoimmune encephalomyelitis (EAE) mouse model. Here, our aim was to build a proteome map of the biological changes that occur during MS at the major onset sites—the brain and the spinal cord. We performed quantitative proteome profiling in five specific brain regions and the spinal cord of EAE and healthy mice with high-resolution mass spectrometry based on tandem mass tags.