2-ethyl-6-methyl-3-hydroxypyridine (EMHP) succinate is the original antioxidant and antihypoxic drug commonly prescribed in Russia. The objective of this study was to develop a rapid, simple and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for EMHP quantitation in rat brain tissue with the use of a bead beating homogenizer. The comparison between two approaches to brain tissue preparation was performed, when spiking the blank brain tissue with EMHP reference standard and internal standard (IS) before and after homogenization step. Chromatographic separation was achieved using Zorbax Eclipse Plus C18 column (1.8 μm, 2.1 × 50 mm) and elution was performed with the mobile phase, consisting of 10 mM of ammonium formate aqueous solution with 0.1% formic acid as solvent A and 0.1% formic acid in methanol as solvent B [44%(А):56%(В), v/v]. Flow rate was 0.4 mL/min and the total run time for each sample analysis was 2.0 min. EMHP and amantadine, IS of this study, were analyzed in positive ionization mode. Ion transitions of m/z 138.0 → 123.0 for EMHP and m/z 152.0 → 135.0 for amantadine were selected in multiple reaction monitoring mode. The developed method for EMHP determination in rat brain samples was validated for selectivity, linearity, accuracy, precision, matrix effects, and stability. The lower and upper limits of quantification were determined to be 1 and 1500 ng/g, respectively. The developed and validated HPLC-MS/MS method was successfully applied to determine EMHP concentrations in rat brain tissue following the intraperitoneal administration at a dose of 3.4 mg/kg.