This study was conducted to identify suitable experimental conditions for the Bovine Corneal Opacity and Permeability (BCOP) assay, after which the assay was employed using 11 substances listed in the OECD test guideline (TG) 437. The opacity of the corneas was checked on days 1, 2, 3, 4, and 10 after extraction. The only day 1 showed the opacity of below 7 (limit value), which indicated the corneas have to be used in a day after extraction. The treatment time of test substances was evaluated at 3, 5, 10, 15, 20, and 30 min to determine the opacity, permeability and IVIS values. Suitable IVIS values were observed at 5, 10, and 15 min. Two culture temperature conditions, 25°C and 32°C, had similar IVIS values. The washing method suggested in the OECD TG 437 resulted in the most suitable IVIS value. Based on the established conditions, the BCOP assay was conducted using the 11 substances listed in OECD TG 437, and the sensitivity, specificity and accuracy matched those in the guidelines for all test substances. Taken together, the results of this study indicate that suitable domestic conditions for BCOP assay as an alternative eye irritation method were established. The results presented herein will be useful to future studies of other Korean alternative researches.