This is the first report of clofazimine (CFZ) penetration and distribution in normal mouse brain. Mice were administered 25 mg/kg CFZ or 100 mg/kg CFZ orally, daily for 2 weeks. Animals were sacrificed and blood and brain tissues were harvested. Liquid chromatography tandem mass spectrometry showed high concentrations of CFZ in homogenized brain, with 100 mg/kg dose having significantly higher concentration than 25 mg/kg. Matrix-assisted laser desorption/ionization spectrometric imaging of brain sections showed widespread tissue distribution of CFZ. Our results show dose dependent localization in brain.