In vivo measurement of multiple neurotransmitters is highly interesting but remains challenging in the field of neuroscience. GABA and L-glutamic acid are the major inhibitory and excitatory neurotransmitters, respectively, in the central nervous system, and their changes are related to a variety of diseases such as anxiety and major depressive disorder. This study described a simple method allowing the simultaneous LC–MS/MS quantification of L-glutamic acid, glutamine and GABA. Analytes were acquired from samples of the prefrontal cortex by microdialysis technique in freely moving mice. The chromatographic separation was performed by hydrophilic interaction liquid chromatography (HILIC) with a core-shell ammonium-sulfonic acid modified silica column using a gradient elution with mobile phases consisting of a 25 mM pH 3.5 ammonium formate buffer and acetonitrile. The detection of L-glutamic acid, glutamine and GABA, as well as the internal standards [d6]-GABA and [d5]-glutamate was performed on a triple quadrupole mass spectrometer in positive electrospray ionization and multiple reaction monitoring mode. The limit of quantification was 0.63 ng/ml for GABA, 1.25 ng/ml for L-glutamic acid and 3.15 ng/ml for glutamine, and the intra-day and inter-day accuracy and precision have been assessed for the three analytes. Therefore, the physiological relevance of the method was successfully applied for the determination of basal extracellular levels and potassium-evoked release of these neuroactive substances in the prefrontal cortex in adult awake C57BL/6 mice.