The grape, its derivatives and byproducts are source of bioactive compounds with high antioxidant activity. The aim of this study was optimize an alternative methodology for evaluate the antioxidant activity using an ex vivo assay thiobarbituric acid reactive substances (TBARS) [1] in liver tissue from healthy mice (registration protocol at CEUA: 001/17 - Faculty of Pato Branco, FADEP). For induction of oxidative stress ex vivo was produced and tested an oxidizing agent - Fe-glutamate complex [2] - easy to obtain, low cost and efficient in physiological pH, being a potential substitute for synthetic oxidant patterns commonly used in this analysis. After optimization of methodology, were evaluated crude extract (GPE) and fractions from red grape pomace obtained by liquid-liquid fractionation with solvents: Hexane (Fr-Hex), Dichloromethane (Fr-CH2Cl2), Ethyl Acetate (Fr-EtOAc) and Ethanol (Fr-EtOH). The extract and fractions were analyzed in concentrations of 375 and 940 mg L-1. The TBARS method is based on the reaction of two molecules of thiobarbituric acid (TBA) with a malondialdehyde (MDA), producing a complex of pink color measured by spectrophotometer at 532 nm. The oxidative stress in the tissue was generated by the release of hydroxyl radicals (•OH) from the reaction between the complex Fe-glutamate with H2O2, as presented in reaction (1). The •OH produced participates in the degradation of biological tissues and in the production of O2•− according reaction (2), which regenerates the Fe-glutamate complex in reaction (3). First were evaluated different concentrations of the oxidizing agent (1; 2; 5; 10; 15 mmol L-1) in mice liver, keeping the concentration of H2O2 constant (65 mmol L-1). Similarly, an antioxidant standard (gallic acid - GA) was tested using the optimal condition of oxidant (5 mmol L-1) previously determined to degrade the tissue. The standard GA (375 and 940 mg L-1) was evaluated and your TBARS’s results were correlated with the results of GPE and fractions (Table 1). The fractions Fr-CH2Cl2 and Fr-EtOAc showed good results for both concentrations tested but at 940 mg L-1 showed better ability to inhibit lipid peroxidation in tissues, resulting in lowest values of malondialdehyde generated per gram of tissue and with no significant difference of gallic acid standard.