Rifampicin (RIF) is a major component for short-course chemotherapy against tuberculosis, since it is active against rapidly metabolizing as well as dormant bacteria. According to the Lipinski rules, RIF should not enter the blood-brain barrier. Visualization of tissue drug distribution is of major importance in pharmacological studies; thus, far imaging of RIF in the brain has been limited to positron emission tomography. We propose using matrix-assisted laser desorption/ionization mass spectrometry imaging techniques as a suitable alternative for the visualization and localization of drug tissue distribution. Using the liquid chromatography mass spectrometric (LCMS) technique, we were able to quantify the concentrations of RIF in the uninfected rat brain; we paired this with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to show the time-dependent manner in which RIF is able to enter the brain. Our results show that even at the minute concentrations measured with LCMS/MS we were able visualize the drug and show its exact distribution in the rat brain. Other available methods require nuclear labeling and the detection of gamma rays produced by labeled compounds to visualize the compound and its localization; MALDI MSI is a more recently developed technique, which can provide detailed information on drug distribution in tissues when compared to other imaging techniques. This study shows that without any requirement for complex preprocessing we are able to produce images with a relatively improved resolution and localization than those acquired using more complex imaging methods, showing MALDI MSI to be an invaluable tool in drug distribution studies.