The purpose of this study was to compare the relative toxicity of a new topical ophthalmic glaucoma medication, travoprost 0.004% without benzalkonium chloride (BAK), with that of commercially available latanoprost 0.005% (preserved with 0.02% BAK) in immortalized human corneal epithelial cells (HCEs). Tissue culture plates (96 well) containing HCEs were divided into 6 groups. Two groups served as negative controls (70% methanol and gentamicin). Another 2 groups—1 in corneal epithelial culture media and the other in a hydroxypropyl (HP)-Guar gellable lubricant eyedrop—served as live controls. The travoprost 0.004% without BAK and latanoprost 0.005% groups were exposed to 100 μL of the undiluted solutions. Cells were incubated for 25 min at 37°C. A live/dead assay was used to measure the effects of travoprost without BAK and of latanoprost on HCEs compared to 70% methanol and culture medium. Between the 2 glaucoma medications tested, travoprost 0.004% preserved without BAK showed significantly less toxicity on HCEs than did latanoprost 0.005%. This difference may have ramifications in terms of tolerability for patients who use these topical glaucoma drugs on a long-term basis.