Energetic failure which occurs in both ischemia/reperfusion and acute drug-induced hepatotoxicity is frequently associated with oxidative stress. This study displays the setting of a new cell culture model for hepatic energetic failure, i.e., HepG2 models modified by etomoxir [ETO] addition [0.1 mM to 1 mM] and compares the cell impact versus tert-butylhydroperoxide [TBOOH; 0.2 mM], an oxidative stress inducer. As it was observed with Minimum Essential Medium (MEM) without any interfering agent, decreasing temperature drastically lowered adenosine triphosphate (ATP) levels, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability test, and protein content, compared to 37 °C (p=0.02, p<0.001 and p<0.001, respectively), but to a larger extent in the presence of ETO or TBOOH. The alteration was generally highly dependent on the ETO concentration, time, and temperature. At 37 °C 24 h after (T24h), regarding ETO concentration, R² correlation ratio was 0.65 (p<0.001), 0.70 (p<0.001), and 0.89 (p<0.001) for ATP levels, protein content, and viability, respectively. The lowest ETO concentration producing a significant effect was 0.25 mM. Concerning time dependency (i.e., T24h versus after 5 h (T5h)), at 37 °C with ETO, ATP level continued to significantly decrease between T5h and T24h. In a similar way, at 37 °C, the MTT viability test decrease was accelerated only between T5h and T24h for ETO concentrations higher than 0.5 mM (p=0.016 and p=0.0001 for 0.75 and 1 mM, respectively). On the contrary, with TBOOH, comparing T24h versus T5h, cellular indicators were improved but generally remained lower than MEM without any interfering agent at T24h, suggesting that TBOOH action was time limited probably in relation with its oxidation in cell medium. This study confirms the interest of altered ETO cell model to screen agents (or formulation) prone to prevent or treat energetic depletion in relation with oxidative stress. © Springer Science+Business Media B.V. 2011