In the therapy of ocular diseases, different active pharmaceutical ingredients are often instilled concomitantly as different ophthalmic solutions at an appropriate interval (unfixed-combination therapy) or simultaneously as one formulation (fixed-combination therapy). In this study, we aimed to compare the in vitro cytotoxicity of fixed- and unfixed-combination ophthalmic solutions using the glaucoma therapeutic agents, timolol and brimonidine. Cultured human corneal epithelial cell line was used as a test system. Exposure period was set at 5 min. Compared with the fixed-combination treatment, the unfixed-combination treatment (timolol followed by brimonidine, and vice versa) led to a significant reduction in the percentage of cell viability. In conclusion, it was suggested that a fixed-combination ophthalmic solution was expected to cause less cellular damage to the ocular surface, compared to an unfixed-combination ophthalmic solution.