We evaluated the in vitro cytotoxicity of benzalkonium chloride (BAK) -containing antiglaucoma eyedrops. We prepared cell cultures of SIRC, BCE C/D-1b, RC-1, and Chang conjunctiva. The viability of cell cultures was determined using the MTT and neutral red assays. The cell viability score (CVS) was used to compare the toxicity of test solutions. %CVS50 and %CVS40/80 of each eyedrop solution were 71 and 26 for Lumigan® (0.002% bimatoprost with 0.005% BAK) , 100 and 99 for Tapros® (0.0015% tafluprost, a new formula from 2010 with 0.001% BAK) , 39 and -29 for 2% Trusopt® (2% dorzolamide with 0.0075% BAK) , 28 and -43 for Xalacom® (latanoprost/0.5% timolol with 0.02% BAK) , 88 and 66 for DuoTrav® (travoprost/0.5% timolol with no BAK) , 36 and -35 for Cosopt® (2% dorzolamide/0.5% timolol with 0.0075% BAK) and 53 and -1 for Combigan® (0.15% brimonidin/0.5% timolol with 0.005% BAK) . Only Xalacom® and Tapros® did not show an apparent decrease in %CVS as compared to the corresponding concentration of BAK. In conclusion, the cytotoxicity of tested eyedrops was dependent on BAK. Only the eyedrops containing latanoprost or tafluprost showed a reduction in the cytotoxicity of BAK.