Tight junction and epithelial barrier disruption is a common trait of many gastrointestinal pathologies, including chemotherapy-induced gut toxicity. Currently, there are no validated in vitro models suitable for the study of chemotherapy-induced mucosal damage that allow paralleled functional and structural analyses of tight junction integrity. We therefore aimed to determine if a transparent, polyester membrane insert supports a polarized T84 monolayer with the phenotypically normal tight junctions. T84 cells (passage 5–15) were seeded into either 0.6 cm², 0.4 µm pore mixed-cellulose transwell hanging inserts or 1.12 cm², 0.4 µm pore polyester transwell inserts at varying densities. Transepithelial electrical resistance was measured daily to assess barrier formation. Immunofluoresence for key tight junction proteins (occludin, zonular occludens-1, claudin-1) and transmission electron microscopy were performed to assess tight junction integrity, organelle distribution, and polarity. Reverse transcription-polymerase chain reaction was performed to determine expression of toll-like receptor 4 (TLR4). Liquid chromatography was also conducted to assess SN38 degradation in this model. Polyester membrane inserts support a polarized T84 phenotype with functional tight junctions in vitro. Transmission electron microscopy indicated polarity, with apico-laterally located tight junctions. Immunofluorescence showed membranous staining for all tight junction proteins. No internalization was evident. T84 cells expressed TLR4, although this was significantly lower than levels seen in HT29 cells ( P = .0377). SN38 underwent more rapid degradation in the presence of cells (−76.04 ± 1.86%) compared to blank membrane (−48.39 ± 4.01%), indicating metabolic processes. Polyester membrane inserts provide a novel platform for paralleled functional and structural analysis of tight junction integrity in T84 monolayers. T84 cells exhibit the unique ability to metabolize SN38 as well as expressing TLR4, making this an excellent platform to study clinically relevant therapeutic interventions for SN38-induced mucosal damage by targeting TLR4.