Fasiglisam (TAK-875) was developed as a first in class, oral, highly potent and selective GPR40 agonist developed for the treatment for type 2 diabetes mellitus. However, during clinical development TAK-875 was terminated in Phase III due to elevated liver safety signals. A human based in vitro multiparametric approach was undertaken to investigate the potential hepatotoxic mechanism of action of TAK-875 with an emphasis of understanding the role of mitochondria in TAK-875 toxicity. Utilising high content screening (HCS) GSH levels, reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) and ATP levels were measured in HepG2 cells, primary human hepatocytes and 3D human liver microtissues (HLiMT) following exposure to TAK-875 over a range of timepoints. In addition mitochondrial function was assessed using the Seahorse XFe96 analyser to determine oxygen consumption rates (OCR) and reserve capacity in HepG2 cells and human hepatocytes following exposure to TAK-875 over a range of timepoints. HCS data showed that TAK-875 in HepG2 cells decreased ATP levels at 24 hours exposure with an AC50 of 104 µM. In human hepatocytes TAK-875 decreased MMP and mitochondrial mass with AC50’s 105 µM and 185µM after 1 hour exposure. At 24 hours exposure the MMP AC50 was 90.4 µM with concurrent decreases in GSH and ATP levels observed. In HLiMT decreases in GSH content and cellular ATP were observed following TAK-875 exposure. The Seahorse analyser data showed that the OCR AC50’s of TAK-875 in HepG2 cells were 16.9, 9.2 and 4.1 µM at 0hr, 1hr and 24hrs respectively. The reserve capacity AC50’s of TAK-875 in HepG2 cells were 12.2, 4.66 and 0.97µM at 0hr, 1hr and 24hrs respectively. In human hepatocytes the OCR and reserve capacity AC50 values at 0hr were determined to be 86.4 and 116 µM. The human plasma Cmax of TAK-875 is 10.1 µM therefore this data demonstrates that early in vitro markers of mitochondrial toxicity were predicted below the Cmax for TAK-875. In vitro screening of TAK-875 preclinically for mitochondrial liability could have predicted the human hepatotoxic effect of TAK-875 and clearly demonstrates the potential role of in vitro screening for hepatotoxicity in drug discovery strategies for mitigating late stage clinical failures.