Nerve injury-induced neuropathic pain is a major public health problem worldwide. Current treatment for neuropathic pain has had limited success because the mechanisms that underlie the induction and maintenance of neuropathic pain are incompletely understood. However, recent advances in proteomics may allow us to uncover complicated biological mechanisms that occur under neuropathic pain conditions. Here, we introduce a combined approach of two-dimensional gel electrophoresis (2-DE) with mass spectrometry (MS) to identify the expression changes in synaptosome-associated proteins in spinal cord dorsal horn after unilateral fifth spinal nerve injury. In 2-DE, a set of highly abundant synaptic proteins with a pI range of 4-7 are separated and compared by size fractionation (25-100 kDa). Then, the differentially expressed proteins are identified and validated by MS, and their potential involvement in physiological and pathological processes is searched. Thus, proteomic analysis can provide expression profiles of synaptic proteins and their posttranslational modifications in cells, tissues, and organs of the nervous system under neuropathic pain conditions.