Epithelial cell monolayers are routinely used to evaluate efficacy of paracellular permeability enhancers (PPEs). The purpose of the present work was to investigate how biorelevant refinements to the Caco-2 cell model impact in vitro efficacy (decrease in transepithelial electrical resistance and increase in mannitol permeability) of PPEs. Standard transport buffer was replaced by fasted-state simulated intestinal fluid (FaSSIF) or serum; or stirring was performed to decrease the unstirred water layer thickness. Apical FaSSIF significantly reduced the efficacy of amphiphilic PPEs palmitoylcarnitine and hexadecylphosphocholine and reduced the amount of these PPEs associated with cells. In contrast, FaSSIF did not affect efficacy of nonamphiphilic PPEs, ethylenediaminetetraacetic acid or 3-nitrocoumarin. Basolateral serum increased the transepithelial flux of PPEs, but did not lessen their potency. Stirring increased the flux of all PPEs, and also enhanced the potency of the amphiphilic PPEs. These results show that inclusion of FaSSIF and agitation in the cellular models significantly alter the efficacy of amphiphilic PPEs but not of hydrophilic or lipophilic PPEs. Future studies should be directed at evaluating the ability to these refined in vitro systems to predict in vivo effects of PPEs.