To determine the cytotoxicity of antibiotic eyedrops to ocular surface cells using a semi-quantitative method, a range of commercially available antibiotic eyedrops were assessed by using three corneal cell lines and one conjunctival cell line. All antibiotic solutions were free of benzalkonium chloride. Cell viability was determined by the MTT assay and neutral red assay following the exposure of cells to the undiluted, 2- and 10-fold diluted drugs for 10, 30, and 60 min. Toxicity was compared using % cell viability score (%CVS) . The tested eyedrops and values of %CVS50 and %CVS40/80 were Bestron(®) (cefmenoxime, 100, 94) , Panimycin(®) (dibekacin, 86, 58) , Noflo(®) (norfloxacin, 90, 50) , Cravit(®) (levofloxacin, 86, 46) , Tosfulo(®) (tosufloxacin, 57, -3) , and Vigamox(®) (moxifloxacin, 57, -6) . Cell viability markedly increased after dilution. For instance, cell viability assayed by MTT was > 80% for all the measurements in antibiotics diluted 10-fold, and the rate of the measurements showing > 80% cell viability decreased to 43% (31 out of 72 measurements) in the solutions diluted 2-fold. Of the drugs tested, Bestron(®) containing cefmenoxime showed the weakest toxicity. Vigamox(®) containing moxifloxacin and Tosuflo(®) containing tosufloxacin were more toxic when compared with the other antibiotics. CVS was useful for the comparison of the cytotoxicity of the drugs.