To prospectively evaluate a new chemical entity’s (NCE) cholestatic DILI potential, an appropriate model system must support the necessary biological processes involved in BA homeostasis while supporting nuclear receptor signaling and drug metabolism. Using Transporter Certified™ sandwich‑cultured human hepatocytes (SCHH), the C-DILI™ assay (patent pending) is a novel predictive cholestatic hepatotoxicity model that evaluates a NCE’s potential to disrupt the BA homeostasis mechanism (e.g. BSEP inhibition, basolateral efflux inhibition, and FXR antagonism). Utilizing this technology, we evaluated the thiazolidinedione class of compounds including troglitazone, pioglitazone, and rosiglitazone to determine the feasibility of using C-DILI™ assay and mechanistic studies (e.g. BSEP inhibition, FXR antagonism) in SCHH to develop structural activity relationships (SAR). The C-DILI™ assay uses media which sensitizes the SCHH system to disruptions of BA homeostasis resulting in LDH leakage. Both pioglitazone and rosiglitazone, under sensitization conditions, showed no change from vehicle control indicating no significant disruption of BA homeostasis. However under these same sensitizing conditions, troglitazone treatment increased LDH leakage in SCHH 792% greater than solvent control. These C-DILI™ assay results were consistent with clinical evidence, indicating that troglitazone has high cholestatic hepatoxicity potential, while pioglitazone and rosiglitazone have low cholestatic hepatotoxicity potential. All three compounds were found to inhibit biliary efflux of d8-TCA in SCHH utilizing B-Clear™ technology. These results indicated that intracellular concentrations of each of the thiazolidinediones and/or metabolites were adequate to inhibit BSEP function. However, troglitazone was the only thiazolidione evaluated in SCHH that blocked FXR activation. Evaluation of the structures of all three drugs shows that each molecule shares a common core structure; however, the α-tocopherol side chain of troglitazone is unique. This case study highlights how the C-DILI™ assay and additional mechanistic studies can be used to perform SAR early in the drug development process to identify key molecular motifs, like the α-tocopherol side chain, to mitigate liabilities toward BA homeostasis and hepatic toxicity.