Differences between male and female brains only seem beginning to be understood with their extreme complication influenced by many intrinsic and extrinsic factors such as genetics, physiological experience, learning procedure, and environment. Regional distribution for the baseline level of brain neurotransmitters in different genders still remains unknown. Thus, it is necessary to establish a reliable analytical method to monitor neurotransmitters and their metabolite levels in brain tissues to elucidate the differences in the pathophysiology of neurological diseases. In this study, a rapid, sensitive and reliable LC-MS/MS method was established for a comprehensive and adequate analysis of neurotransmitters and their metabolites, including γ-aminobutyric acid (GABA), glutamate (Glu), serotonin (5-HT), 5-Hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), acetylcholine (Ach), choline (Cho), dopamine (DA) and 3,4-Dihydroxyphenylacetic acid (DOPAC) in various brain regions including the motor cortex, nucleus accumbens, somatosensory cortex, corpus striatum, medial prefrontal cortex, thalamus, hypothalamus, hippocampus cornu ammonis (CA), rhinal cortex, auditory cortex, visual cortex, superior colliculus, olfactory bulb, substantia nigra, dorsal raphe and locus coeruleus between male and female mice brain tissues.