Background. Products released by neutrophils are partly responsible for the destruction observed in periodontitis. We describe the validation of a rapid, non-invasive oral rinse assay to enable the accurate quantification of oral neutrophils. Methods. 53 patients with periodontitis and 14 controls provided rinse samples prior to and following phase I treatment. Cells in the samples were stained, and counts were carried out using a hemocytometer. Results. A significant difference in pretreatment oral neutrophil counts was observed between those with periodontal disease and the healthy controls. The severe disease group had more than five times the number of neutrophils as the healthy controls (P < 0.001). Patients who responded favorably to therapy demonstrated a 43% reduction in neutrophil counts. Patients who did not respond to treatment showed no difference in neutrophil levels. Conclusion. This assay is an effective means of collecting and quantifying neutrophils for the purpose of monitoring disease treatment response.