The present study aimed to investigate the effect of serum derived from advanced platelet-rich fibrin on the healing of experimentally-induced corneal ulceration in donkeys. Nine adult apparently healthy donkeys of both sexes were used after being proofed normal after ophthalmological examination. A 6-mm-diameter centric corneal ulceration was induced chemically by using 1N sodium hydroxide solution. The donkeys were then randomly allocated into two main groups; control group (A), in which the corneal ulcer left for the period of the study without application of medicaments, and group (B), in which serum derived from advanced platelet-rich fibrin (s-PRF-drops) was applied to the eye three times/day for 35 days. Evaluation criteria included; clinical and external ophthalmic examinations, fluorescein staining, ulcer healing by analyzing serial digital photographs and histopathology. Results revealed no significant changes in the evaluation criteria between treatment and control groups. Corneal ulcer healing was associated with corneal opacity, vascularization, melanosis, and other complications that likely negate any potential benefit of administration of s-PRF as a treatment for corneal ulcer in donkeys. Histological results of the s-PRF-drops group were similar to the other group in the degree of re-epithelialization and regularity of the collagen bundles, type and maturity of the collagen. However, treatment by s-PRF drops resulted in no epithelial keratinization and slightly less sub-epithelial stromal inflammatory reaction.