Although there are various drugs for Neuropathic pain (NP), the effects of single drugs are often not very satisfactory. The analgesic effects of different combinations of pregabalin, duloxetine, and tramadol or the combination of all three are still unclear. Mixtures of two or three drugs at low and high concentrations (7.5, 10, 15, and 20 mg/kg pregabalin; 7.5, 10, 15, and 30 mg/kg duloxetine; 5 and 10 mg/kg tramadol) were administered to chronic postischemic pain (CPIP) and spinal nerve ligation (SNL) model mice. The effects of these combinations of drugs on mechanical allodynia were investigated. The expression of the glial fibrillary acidic protein (GFAP) in the spinal cord and dorsal root ganglia (DRGs) was measured. The combination of pregabalin, duloxetine, and tramadol significantly alleviated mechanical hyperalgesia in mice with CPIP and SNL. After the administration of this drug combination, the expression of GFAP in the spinal cord and DRGs was lower in the CPIP and SNL model mice than in control mice. This result suggests that the combination of these three drugs may be advantageous for the treatment of NP because it can reduce side effects by preventing the overuse of a single drug class and exert increased analgesic effects via synergism.