The expression pattern of proteins defines the range of biological processes in cellular subcompartments. A core aim in cell biology is therefore to determine the localization and composition of protein complexes within cells. Proximity labeling methodologies offer an unbiased and efficient way to unravel the cellular micro-environment of proteins, providing insights into the molecular networks they participate in. In this chapter, we present a protocol for conducting proximity labeling experiments in primary murine neuronal cultures in vitro based on the proximity-dependent biotinylation identification (BioID) approach. Data acquired through this protocol can be utilized to identify the composition of protein complexes in neurons and to create molecular maps of neuronal subcompartments. This will aid in determining the spatial distribution of biological processes within neurons, and in unraveling fundamental principles of neuronal function and plasticity.