A prerequisite for successful oral drug therapy is the drug's ability to cross the gastrointestinal barrier. Considering the increasing number of new chemical entities in modern drug discovery, reliable and fast in vitro models are required for early and efficient prediction of intestinal permeability. To mimic the intestinal environment, use of biorelevant media may provide valuable information on in vivo drug permeation. The present study aims at improving the novel biomimetic phospholipid vesicle-based permeation assay's (PVPAbiomimetic) biorelevance by investigating the applicability of the biorelevant media; fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF). The FaSSIF and FeSSIF's influence on the permeability of the model drugs acyclovir, indomethacin, griseofulvin and nadolol was then assessed. The barriers' robustness in terms of storage stability was also evaluated. The barriers were found to maintain their integrity in presence of FaSSIF and FeSSIF. The model drugs showed changes in permeability in presence of the different simulated intestinal fluids that were in agreement with previous reports. Moreover, the barrier showed improved storage stability by maintaining its integrity for 6months. Altogether, this study moves the PVPAbiomimetic an important step towards a better in vitro permeability model for use in drug development.