Purpose: To determine the stability of bimatoprost, latanoprost, and travoprost under conditions of simulated daily use and varying degrees of thermal stress. Methods: Commercially available bimatoprost, latanoprost, and travoprost were obtained in their original bottles as distributed by the manufacturers. Bottles were stored in calibrated, nonhumidified, light-free incubators maintained at 278C, 378C, or 508C for 3, 9, 15, or 30 days. Capped bottles were inverted and left uncapped for 1 min daily to simulate patient use; no drops were expelled. Bimatoprost concentration was analyzed using liquid chromatography with ultraviolet detection at 210 nm. Latanoprost and travoprost concentrations were analyzed by liquid chromatography/tandem mass spectrometry (MS/MS) using selected reaction monitoring. Results: Off-the-shelf control bottles of bimatoprost contained 102% of the labeled concentration. In all combinations of stress temperature and duration, mean bimatoprost concentration ranged from 100% to 116% of the labeled concentration with no measurable degradation. Off-the-shelf control bottles of latanoprost contained 115% of the labeled concentration. Mean latanoprost concentration ranged from 97% to 120% of the labeled concentration. Latanoprost was stable at 278C. When stressed at 378C or 508C, latanoprost degraded at a rate of 0.15 or 0.29 mg/mL/day, respectively. Off-the-shelf control bottles of travoprost contained 120% of the labeled concentration. Mean travoprost concentration ranged from 83% to 142% of the labeled concentration. Travoprost was stable at 278C and 378C, although concentration measurements at 378C exhibited high variability. When stressed at 508C, travoprost degraded at a rate of 0.46 mg/mL/day. Conclusions: Higher than expected concentrations for stressed drug samples are likely a result of evaporation. Under the conditions of thermal stress tested in this study, bimatoprost remained stable for all conditions tested. Latanoprost degradation was measurable only in samples stressed at 378C and 508C, whereas travoprost degradation was statistically significant only in samples stressed at 508C.