Microphysiological systems that can accurately represent in vivo parameters have been recently developed as alternatives to animal testing. However, representative toxicity testing requires that absorptive, distributive, metabolic, and excretion (ADME) characteristics of microphysiological systems are characterized to ensure that they closely mimic those in vivo. The present study employed the Mimetas OrganoPlate® 3-lane tissue chip microphysiological system and traditional transwell techniques to study intestinal permeability of four drugs. The Mimetas model is comprised of two microfluidic channels separated by a gel barrier. Caco-2 cells were seeded in one channel. The transwell system contains two chambers separated by a membrane barrier. In this system, Caco-2, and human intestinal enteroid cells isolated from jejunal (J2 isolate) and duodenal (D109 isolate) tissues of adult patients were seeded inside the transwell. Caffeine (1, 10, or 100 µM), fexofenadine (4, 40, or 400 µM), indomethacin (2, 20, or 200 µM) or propranolol (0.1, 1, or 10 µM) were added to the apical or basolateral chambers. In both systems, the compounds were allowed to diffuse from the donor to the recipient channel (Mimetas) or chamber (Transwell) over 100 minutes or 15 hours. Media was collected from both sides, concentrations were determined using liquid chromatography tandem mass spectrometry, and apparent permeability was quantified for each condition. We found that it is difficult to establish mass balance in the Mimetas system for tested compounds due to considerable retention of the test substances in the chip’s gel layer. These observations indicate that use of the Mimetas 3-lane model for ADME studies may be limited, as permeability of compounds in this model is a function of both the cell-enabled physiological barrier and the artificial gel barrier. Additionally, apparent permeability was generally lower in the transwell model using the enteroid cell types. Overall, this study highlights the importance of comprehensive evaluation of new models as alternative approaches to toxicity testing. Future studies will investigate the effects of flow on transport kinetics using the CN-Bio PhysiomimixTM OOC system.