Evaluation of ocular hyperemia has been an important assessment in research studies of effects of contact lenses, medications, and pollutants on the eye. Hyperemia has been difficult to quantitate objectively. The purpose of this study was to validate a computer based image analysis system to quantitate hyperemia automatically and objectively in pixelated images of the external eye using two measures, the percent of the red color, RR, and the fraction of pixels which are blood vessels, VA. Validation was against an established photographic reference scale of ocular hyperemia and against the clinical pharmacologic effects of 0.5% dapiprazole hydrochloride, known to increase hyperemia, and 2.5% phenylephrine hydrochloride, known to decrease hyperemia. Color transparencies from the reference scale were converted to digital images. Temporal and nasal regions of the external eye were imaged directly to magnetic disk before and after pharmacologic intervention. Custom software automatically excluded unwanted regions, and quantitative image analysis produced RR and VA. RR and VA were each correlated with the reference scale. For each region and for each pharmacologic intervention, the mean RR and the mean VA, respectively, were compared at time zero and at a mean elapsed time of 713 +/- 47 s. RR and VA consistently increased as the hyperemia in the reference scale increased. Pearson correlation coefficients were 0.98 and 0.99, respectively, (p < 0.01). At 713 +/- 47 s after each pharmacologic intervention, RR and VA increased and decreased as expected (p < 0.001). Thus, this study successfully validated the methodology against expert clinical judgment and was able to measure automatically and objectively clinical changes in ocular hyperemia.