Introduction: Immunohistochemical (IHC) analysis of stained sections of vestibular specimens have been used to define diagnostic criteria for neuroproliferative vestibulodynia (NPV). Bornstein’s criteria (2004) included manually counting 8 or more mast cells in a standardized microscopic field and/or subepithelial nerve fibers that were 10 times higher than controls. However, there is wide variability in manual counting, IHC staining and magnification. We examined a more reproducible methodology by assessing percentage of immunopositive stained area using computer-assisted histometry. Methods: Samples from 17 patients were stained with CD117, a marker of mast cells, and PGP9.5, a neuronal marker. Photomicrographs were obtained using 200x and 400x magnification and analyzed using Image J with 3 measurements per photomicrograph. This methodology was also applied to previously published images of tissues from patients with and without NPV (Tympanidis 2003; Goetsch 2010). For comparison, CD117-immunopositive cells were also manually counted, as described by Bornstein. Clusters of immunopositive granules positively stained and separated from an adjacent cell membrane were counted as a single immunopositive cell. Results: Using Image J, mean CD117-immunopositive area was 2107 ± 1339 µm2, representing 0.79 ± 0.51% of total area analyzed. Mean PGP9.5-immunopositive area was 1123 ± 695 µm2, representing 0.38 ± 0.27% of total area analyzed. Using manual counting, we observed 30.6 ± 15.3 CD117-immunopositive cells per standardized field (200x), greater than the Bornstein threshold of 8 mast cells per standardized field. Tissues from control patients without vestibulodynia had 6 to 8-fold lower immunostained area than tissues from vestibulodynia patients using computer-assisted histometry. In analyses of photomicrographs from Goetsch and Tympanidis, 2.48% and 2.87% of the control areas were immunopositive. For controls, using Image J, immunostained area for PGP 9.5 was 2.6-fold greater than that reported by Bornstein using manual counting (425 µm2). Conclusions: All our patients had increased density of CD117-immunopositive cells and increased PGP9.5-immunopositive area, confirming their NPV diagnosis. To facilitate more consistent, widespread and rapid assessment of IHC analysis of stained sections of vestibular specimens, we propose using computer-assisted histometry for future studies. SOURCE OF Funding: None