Abstract: Conjunctival hyperemia is observed in a variety of ocular inflammatory conditions. The evaluation of hyperemia is indispensable for the treatment of patients with ocular inflammation. However, the major methods currently available for evaluation are based on nonquantitative and subjective methods. Therefore, we developed novel software to evaluate bulbar hyperemia quantitatively and objectively. First, we investigated whether the histamine-induced hyperemia of guinea pigs could be quantified by image analysis. Bulbar conjunctival images were taken by means of a digital camera, followed by the binarization of the images and the selection of regions of interest (ROIs) for evaluation. The ROIs were evaluated by counting the number of absolute pixel values. Pixel values peaked significantly 1 minute after histamine challenge was performed and were still increased after 5 minutes. Second, we applied the same method to antigen (ovalbumin)-induced hyperemia of sensitized guinea pigs, acquiring similar results except for the substantial upregulation in the first 5 minutes after challenge. Finally, we analyzed human bulbar hyperemia using the new software we developed especially for human usage. The new software allows the automatic calculation of pixel values once the ROIs have been selected. In our clinical trials, the percentage of blood vessel coverage of ROIs was significantly higher in the images of hyperemia caused by allergic conjunctival diseases and hyperemia induced by Bimatoprost, compared with those of healthy volunteers. We propose that this newly developed automated hyperemia analysis software will be an objective clinical tool for the evaluation of ocular hyperemia.