AIM: To develop an image-based objective method to precisely evaluate regional ocular bulbar injection.METHODS:Six healthy adult volunteers were photographed in four orientations (superior, inferior, nasal and temporal sides) with and without stimulating eye drops. Six line segments (covering 30°) were drawn 4mm away from the limbus on each image using ImageJ software. The graph peaks, which were derived from the areas under the line segments and corresponded to the cross-sectional grey-level of the vessels, were analyzed to obtain peak area, peak height/width (PH/PW), and peak numbers. Different-sized areas were selected to calculate the pixels based on the edge-detection algorithm. Also, conjunctival and superficial scleral vessels were analyzed separately. RESULTS:This method had a smaller coefficient of variation, especially for PH/PW, in all four orientations. Hyperaemia parameters changed the least after challenging in the superior region. Moreover, 95% of the PH/PW ratios were greater than 0.87 in conjunctival vessels and less than 1.00 in superficial scleral vessels. PH/PW significantly increased in conjunctival vessels and changed less in superficial scleral vessels.CONCLUSION:A new method of objectively assessing bulbar injection based on ocular surface images was developed. This method can be used to quantify ocular regional injection and to distinguish the superficial scleral and conjunctival vessels.