Gβγ dimers are one of the essential signaling units of activated G protein-coupled receptors (GPCRs). There are five Gβ and 12 Gγ subunits in humans; numerous studies have demonstrated that different Gβ and Gγ subunits selectively interact to form unique Gβγ dimers, which in turn may target specific receptors and effectors. Perturbation of Gβγ signaling can lead to impaired physiological responses. Moreover, previous targeted multiple-reaction monitoring (MRM) studies of Gβ and Gγ subunits have shown distinct regional and subcellular localization patterns in four brain regions. Nevertheless, no studies have quantified or compared their individual protein levels. In this study, we have developed a quantitative MRM method not only to quantify but also to compare the protein abundance of neuronal Gβ and Gγ subunits. In whole and fractionated crude synaptosomes, we were able to identify the most abundant neuronal Gβ and Gγ subunits and their subcellular localizations. For example, Gβ1 was mostly localized at the membrane while Gβ2 was evenly distributed throughout synaptosomal fractions. The protein expression levels and subcellular localizations of Gβ and Gγ subunits may affect the Gβγ dimerization and Gβγ-effector interactions. This study offers not only a new tool for quantifying and comparing Gβ and Gγ subunits but also new insights into the in vivo distribution of Gβ and Gγ subunits, and Gβγ dimer assembly in normal brain function.