Background and Purpose: Intestinal absorption is a critical factor in characterizing orally administered drugs. A well-established method for the assessment of intestinal pharmacokinetics involves the use of Caco-2 cells cultured on Transwell inserts. However, this model does not fully capture the roles of the different gut segments that play a role in differences of intestinal absorption of oral drugs. Methods: In this study, we used a panel of 14 reference compounds, using a concentration-response design, in three human segment-specific intestinal cell types: Caco-2 cells, and jejunal (J2) and duodenal (D109) enteroid-derived cells. The 14 tested compounds were selected for their known differences in absorption and toxicity within the gastrointestinal system (Benzophenone-4, Streptomycin, Buspirone, Nefazodone, Chlorpromazine, Gentamycin, Tenofovir Disoproxil Fumarate, Indomethacin, Ibuprofen, Sulforaphane, Perfluorooctane sulfonate, Staurosporine, Afatinib, and Idarubicin). Cells were seeded in 96-well Transwell plates to accommodate high-throughput testing of compounds, concentrations, and replicates. Cytotoxicity was assessed using lactate dehydrogenase (LDH) activity, and transepithelial electrical resistance (TEER) was measured to evaluate barrier integrity. Samples were collected at 2-, 24-, and 48-hours post-treatment, and drug transport was quantified using mass spectrometry. Results: TEER data revealed that Caco-2 cells were the most responsive to gut-specific toxic compounds (Staurosporine, Afatinib, and Idarubicin). These compounds disrupted TEER in both J2 and D109 cells after 48 hours of exposure at the highest concentrations. In contrast, LDH assays indicated that Caco-2 cells were less sensitive, while J2 and D109 cells exhibited cytotoxic responses to many of the tested compounds. Analysis of intestinal permeability data showed a broad range of absorption across the tested compounds, with segment-specific differences observed. For instance, Indomethacin was absorbed at the highest rate by J2 cells, aligning with known human data that show its primary absorption occurs in the small intestine rather than the colon. Similar results were observed for Ibuprofen, again aligning with known clinical data. Conversely, Benzophenone-4, a polar and highly water-soluble compound, showed very low absorption in all tested cell types. Conclusions: In summary, this study demonstrates the utility of intestinal segment-specific in vitro models for studying pharmacokinetics and toxicity. Our findings suggest that LDH is a sensitive marker of cytotoxicity in human intestinal enteroid-derived cells, while TEER is a more sensitive measure of toxicity for Caco-2 cells. Moreover, the observed differences in drug absorption among segment-specific cell types provide valuable data that can enhance the accuracy of in silico models of drug pharmacokinetics.