The central nervous system contains a complex intermingled network of neuronal, glial, and vascular cells, and for several decades, neurobiologists have used subcellular fractionation methods to analyze the molecular structure and functional features of the different cell populations. Biochemists have optimized fractionation protocols that enrich specific compartments such as synapses (called “synaptosomes”) and synaptic vesicles to reduce this complexity. However, these approaches suffered from a lack of specificity and purity, which is why we previously extended the conventional synaptosome preparation to purify fluorescent synaptosomes from VGLUT1venus knock-in mice on a cell sorter. We adapted our previous protocol to sort from single neuronal projections and small target regions of the brain as we did in the present example by labeling dopaminergic projections to the striatum. We proved that our newest method allows a steep enrichment in fluorescent dopaminergic synaptosomes containing presynaptic varicosities and associated postsynaptic elements and a substantial depletion in glial contaminants. Here we propose a detailed procedure for implementing projection-specific fluorescence-activated synaptosome sorting.