Per- and polyfluoroalkyl substances (PFAS) are a broad class of chemicals with high environmental persistence and growing ecological and human health concern. Perfluorooctane sulfonate (PFOS) is among the most detected PFAS in environmental sampling and is often at high concentrations and has been measured in a variety of ecological receptors from around the globe. Despite the prevalence of PFOS, there are still important data gaps with regard to toxicity. For example, while studies have confirmed PFOS accumulation in tissues of wild reptiles, reptilian laboratory toxicity data are lacking. The purpose of this study was to first develop toxicity testing protocols using the brown anole, Anolis sagrei and, second, to conduct PFOS dosing studies to generate toxicity data to support ecological risk assessment of reptiles. We conducted two studies in which subadult males and then mature males were dosed with PFOS via pseudo-gavage with maximum doses in the range of 2 mg/kg/d. Subadults were exposed for 35 days while adult males were exposed for 90 days. While no significant mortality was observed, subadult male size and growth rate were significantly affected by PFOS at 2.98 mg/kg/d with a corresponding no effect level of 0.20 mg/kg/d and corresponding 10 and 20% effect levels for growth rate of 0.22 and 0.44 mg/kg/d, respectively. There were no significant effects on size metrics of mature male anoles from the 90-day study although several metrics showed dose-dependent decreases. These data may serve as the basis for toxicity reference values (TRVs) for use in ecological risk assessments of PFAS-contaminated sites in which reptiles may be exposed.