Objective: The quantitative analysis of the changes of the in situ GABA-immunoreactivity in the dorsal horn of the spinal cord (SDH) remains challenging. Therefore, we introduced a novel evaluation technique based on immunohistochemistry images. Study Design: Eight Sprague-Dawley rats underwent a spinal cord dorsal hemisection, and we induced chronic neuropathic pain (CNPSCI). Immunofluorescent staining with markers of NeuN and GABA was performed. We developed a method with an ellipse region of interest (ROI) and baselines according to the distribution of NeuN-positive neurons. We investigated the distribution of the marked cells (NeuN+/GABA+ GABAergic neurons) in each SDH and the immunoreactive intensities of the staining marker (GABA), without the description of the spinal cord laminae. Results: This method showed a significant (p<0.05) loss of GABAergic neurons in the deeper regions on the injured side of the SDH. This was associated with the decrease of GABA immunoreactivity in the chronic phase of CNPSCI (8 weeks after spinal cord injury). Conclusion: This method could normalize the morphometric variations and the deformations in the SDHs in pairs in each transverse-section sample. It allowed us to quantitatively compare the number and the distribution of marked cells in the SDHs and the average immunoreactivities in this ellipse ROI. © The Author(s), 2021.