Troglitazone was introduced as an anti-diabetic drug in the late 1990s, and represented the first of the thiazolidinedione (TZD) class of drugs to be marketed. Troglitazone works by activating the peroxisome proliferator-activated receptor γ (PPARγ), reducing blood glucose levels by enhancing insulin sensitivity, primarily in the adipose and skeletal muscle tissues. Due to increasing concerns of troglitazone-linked hepatotoxicity, troglitazone was withdrawn from the U. K. market in 1997 and U. S. A. in 2000. A considerable effort to elucidate the mechanisms of troglitazone-induced hepatotoxicity has been made, resulting in a variety of proposed mechanisms; formation and accumulation of toxic metabolites, oxidative stress, mitochondrial dysfunction, and inhibition of bile salts excretory protein leading to cholestasis. The purpose of this study was to investigate whether a metabolomics approach can be used to further elucidate the mechanism of toxicity of troglitazone. Furthermore, metabolomic profiling was undertaken to determine if this approach can distinguish between TZDs with hepatotoxic potential (i. e. troglitazone) and those with none (i. e. rosiglitazone and pioglitazone). Conventional MTT assay revealed that troglitazone was a more potent toxin to the HuH-7 hepatoma cell line compared to rosiglitazone and pioglitazone, with an IC50 value of 25.9 +- 1.8, > 168.9 +- 30.0, and > 79.4 +- 13.5 μM, respectively. A comparison between the IC50 values obtained by the MTT versus LDH assays (25.9 +-1.8 versus > 42.7 +- 3.6 μM) suggests that troglitazone affected the reductive capacity of mitochondria at lower level before causing the cells to rupture and release their intracellular contents. An untargeted and a targeted metabolomics approach revealed that troglitazone decreased the levels of intracellular glutamate, malate, energy production, GSH, and nicotinamide at concentrations that are non-toxic by the MTT assay. These observations demonstrate the discriminative ability of a metabolomics approach for the assessment of drug toxicity. The findings also suggest that troglitazone affects the tricarboxylic acid (TCA) cycle, interrupting the anaplerotic and cataplerotic balance of the TCA cycle, leading to oxidative stress and reduced energy level.