Abstract The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice produces a reliable and selective degeneration of the nigrostriatal pathway, a hallmark feature of Parkinson’s disease (PD). Determining the brain concentrations of 1-methyl-4-phenyl pyridium (MPP +), the neurotoxic metabolite of MPTP, is critical for evaluating drugs designed to potentially treat PD. We have developed sensitive and specific quantitative methods for the determination of MPP + in mouse striatal tissue by liquid chromatography/tandem mass spectrometry. The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer. Neutralizing the brain sample with 0.2 M phosphate buffer successfully solved a high-performance liquid chromatography (HPLC) peak tailing of MPP + in brain extracts with 0.4 M perchloric acid (HClO 4) under the reversed phase HPLC conditions, which significantly improved the sensitivity of the method. The HPLC peak shape of MPP + using cation exchange chromatography was not affected by the pH of the samples. Optimization of electrospray ionization (ESI) conditions for the quaternary ammonium compound MPP + established the limits of detection (LOD) (S/N = 3) at 0.34 pg/mg tissue and 0.007 pg/mg tissue (5 μl of injection) using the reversed phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the cation exchange LC/MS/MS, respectively. Both methods were selective, precise (%R.S.D. < 6%), and sensitive over a range of 0.001–1 ng/mg tissue. The cation exchange method showed greater sensitivity and tolerance to low pH samples than the reversed phase method. The developed methods were applied to monitoring changes in MPP + concentrations in vivo. Two reference agents, R-(−) Deprenyl and MK-801, known to alter the concentration of MPP + in MPTP treated mice were evaluated.