We evaluated the cytotoxic effects of four prostaglandin analogs (PGAs) used to treat glaucoma. First we established primary cultures of conjunctival stromal cells from healthy donors. Then cell cultures were incubated with different concentrations (0, 0.1, 1, 5, 25, 50 and 100%) of commercial formulations of bimatoprost, tafluprost, travoprost and latanoprost for increasing periods (5 and 30 min, 1 h, 6 h and 24 h) and cell survival was assessed with three different methods: WST-1, MTT and calcein/AM-ethidium homodimer-1 assays. Our results showed that all PGAs were associated with a certain level of cell damage, which correlated significantly with the concentration of PGA used, and to a lesser extent with culture time. Tafluprost tended to be less toxic than bimatoprost, travoprost and latanoprost after all culture periods. The results for WST-1, MTT and calcein/AM-ethidium homodimer-1 correlated closely. When the average lethal dose 50 was calculated, we found that the most cytotoxic drug was latanoprost, whereas tafluprost was the most sparing of the ocular surface in vitro. These results indicate the need to design novel PGAs with high effectiveness but free from the cytotoxic effects that we found, or at least to obtain drugs that are functional at low dosages. The fact that the commercial formulation of tafluprost used in this work was preservative-free may support the current tendency to eliminate preservatives from eye drops for clinical use.