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Metabolomics approach requires to be optimized for each biological matrix used.At present, no method standardization for rodent brain metabolomics.Designing of methodology for each pre-analytical step (tissue lysis, extraction…).Complementary information on rat brain metabolome using NMR, LC–MS, GC–MS.We developed a multi-platform approach for the metabolome exploration of rat brain tissue, using liquid chromatography coupled with mass spectrometry (LC–MS), nuclear magnetic resonance spectroscopy (NMR) and gas-chromatography coupled with mass spectrometry (GC–MS). The critical steps for metabolite exploration of cerebral tissues are tissue lysis and metabolites extraction. We first evaluated the impact of freeze-drying compared to wet tissue metabolites extraction using NMR and LC–MS with a reversed phase liquid chromatography. Then, we compared four metabolite extraction methods Based on the number of metabolites extracted, their intensity and their coefficient of variation (%CV), the most reproducible protocol (one-step extraction with acetonitrile on lyophilized material) was chosen to further evaluate the impact of sample mass on method performance (3, 6, and 9 mg were essayed). GC–MS analysis was also investigated by analyzing four different methoximation/silylation derivatization combinations. The optimal analytical protocols were proposed to establish the reliability required to realize untargeted brain tissue metabolomics exploration. The most reliable workflow was then exemplified by analyzing three rat brain regions (cerebellum, frontal and parietal cortices, n = 12) by 1H NMR, LC–MS and GC–MS, allowing their clustering based on their metabolic profiles. We present here an example of development of methodology that should be done before running analysis campaigns.