Monoamine oxidases (MAO) catalyze the breakdown of monoamine eurotransmitters. MAO-A metabolizes serotonin and norepinephrine. Deficiency of these two neurotransmitters is associated with the development of depression. Oxidative deamination catalyzed by MAO-B is one of the major pathways of dopamine degradation in the human brain. MAO-B is therefore a target for the treatment of Parkinson’s disease (PD). Herein we present a novel strategy to evaluate MAO activity on mice brains after the systemic administration of MAO  nhibitors. After the treatment with a MAO inhibitor, mice were killed by decapitation. Whole brains were rapidly removed, homogenized and centrifuged. The ex vivo effects of the MAO inhibitor on each treated mice brain homogenate were evaluated in a fluorimetric assay by their effects on the production of hydrogen peroxide (H2O2), from benzylamine (for MAO B) or serotonin (for MAO A) or p-tyramine (for both isoforms) by the corresponding enzyme. H2O2 was detected using Amplex Red reagent in the presence of horseradish peroxidase, where resorufin, a fluorescent product, is produced at stoichiometric amounts. By using this procedure we were able to evidence the ex vivo MAO A and MAO B inhibition by a reference drug, pargyline (MAO B inhibitor) and also by novel compounds developed in our laboratories, even with acute (i.p., oral gavage) or chronical treatment (i.p.). Similar grade of MAO inhibition was observed with the inhibitors systemically administered in mice, and when the inhibitor was added directly into the well. This novel procedure is a useful technique for the development and discovery of new drugs for the treatment of MAO related disorders, such as depression, PD, among others. It allows to test the effect of different compounds on brain MAOs, and to evaluate their ability to cross the blood brain barrier at same time.