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. Here, we evaluate the potential of a two-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. HPLC analysis revealed that the bioaccessibility of fluoxetine in the avian in vitro models (75.9% and 78.6%) was statistically significantly lower than in the mammalian models (88.2-89.6%) as a percentage of what was recovered; however there were no statistically or biologically significant inter-species difference in terms of the amount recovered per gram of 'food' inserted at the start of the simulation. Nevertheless, this in vitro model provides a useful method of comparing the bioaccessibility of pharmaceuticals in different prey for species with different gastrointestinal conditions. There may be merit for ecological risk assessments in further developing this in vitro approach to improve estimates of internal exposure for organics. This article is protected by copyright. All rights reserved.