Abstract   Wildlife can be exposed to human pharmaceuticals via prey that have accumulated the compounds from wastewater, surface water, sediment, and soil. One factor affecting internal absorption of pharmaceuticals is bioaccessibility, the proportion of the compound that enters solution in the gastrointestinal tract. Currently, the bioaccessibility of most pharmaceuticals in prey remains unknown for most wildlife species. The authors evaluated the potential of a 2-compartment in vitro gastrointestinal tract model to compare the bioaccessibility of the antidepressant fluoxetine from invertebrate prey for birds and mammals. Samples of gizzard (or stomach) and intestinal-phase digestive juices were obtained from the in vitro models along with the residual solid material. High-performance liquid chromatographic analysis revealed that the bioaccessibility of fluoxetine in the avian in vitro models was statistically significantly lower than that in the mammalian models as a percentage of what was recovered; however, there were no statistically or biologically significant interspecies difference in terms of the amount recovered per gram of “food” inserted at the start of the simulation. This in vitro model provides a useful method of comparing the bioaccessibility of pharmaceuticals in different prey for species with different gastrointestinal conditions. There is merit for ecological risk assessments in further developing this in vitro approach to improve estimates of internal exposure for organics. Environ Toxicol Chem 2016;35:2349–2357. © 2016 SETAC