Abstract Background Abnormal activation of protease activities during experimental autoimmune encephalomyelitis (EAE) in rats, a rodent model of multiple sclerosis, have been implicated in either the direct destruction of myelin components or the intracellular signal transduction pathways that lead to lymphocyte infiltration, oligodendrocyte destruction, neuronal dysfunctions and axonal degeneration. The identification of changes in regulated proteolytic events during EAE is crucial for uncovering activated proteases that may underline the pathological features such as inflammation and demyelination. We searched for either non-tryptic or semi-tryptic peptides from a previous shotgun proteomics study using isobaric tags for relative and absolute quantification (iTRAQ) to compare the proteomes of normal and EAE rat lumbar spinal cords. Results We discovered that several proteins, such as α1-macroglobulin, a protease inhibitor, α1B-glycoprotein, β2-microglobulin, neurofilament light polypeptide and sulfated glycoprotein 1 had non-tryptic peptide iTRAQ ratios that were substantially different from the overall protein iTRAQ ratios, suggesting that such peptides may be markers for the proteolytic products generated by the protease(s) altered during EAE. Indeed, subsequent Western blotting confirmed the dysregulation of specific protein cleavages in EAE tissues. Additional proteolytic changes in α2-macroglobulin, another protease inhibitor similar to α1-macroglobulin was also observed. Conclusion The results from this study revealed changes among both neuronal protein processing and endogenous proteolysis modulators in EAE animals. This information may provide a rationale for protease inhibitor-based therapeutic interventions for multiple sclerosis.