Optimization of benzalkonium chloride (alkyl dimethylbenzylammonium chloride: BAK) concentration as preservative in 0.0015% tafluprost ophthalmic solution (Tapros 0.0015% ophthalmic solution), an anti-glaucoma medicine, was examined from the points of ocular surface safety and preservative efficacy. BAKC(12), which is dodecyl dimethylbenzylammonium chloride, and BAKmix, which is the mixture of dodecyl, tetradecyl and hexadecyl dimethylbenzylammonium chloride were used in this study. The effects of BAKC(12) concentrations and the BAK types, BAKC(12) and BAKmix, in tafluprost ophthalmic solution on ocular surface safety were evaluated using the in vitro SV 40-immobilized human corneal epithelium cell line (HCE-T). Following treatments of Tafluprost ophthalmic solutions with BAKC(12), its concentration dependency was observed on cell viability of HCE-T. The cell viability of HCE-T after treatment of these solutions with 0.001% to 0.003% BAKC(12) for 5 minutes were the same level as that after treatment of the solution without BAK. Tafluprost ophthalmic solution with 0.01% BAKC(12) was safer for the ocular surface than the same solution with 0.01% BAKmix. Preservatives-effectiveness tests of tafluprost ophthalmic solutions with various concentrations of BAKC(12) were performed according to the Japanese Pharmacopoeia (JP), and solutions with more than 0.0005% BAKC(12) conformed to JP criteria. It was concluded that 0.0005% to 0.003% of BAKC(12) in tafluprost ophthalmic solution was optimal, namely, well-balanced from the points of ocular surface safety and preservative efficacy.