Neurons and astrocytes interact in the brain to form a complex network with physical, chemical, and electrical connectivity. Traditional methods for investigating proteomes have primarily employed either primary cell cultures or cells acutely separated from tissue and purified based on cell surface markers. However, these methods often compromise the cellular physiological integrity, leading to loss of associated proteins and native connectivity. Therefore, to understand the role of astrocytes and their interactions with neurons at the protein level, we designed a cell-specific method using the biotin ligase BioID2, which enables tagging of proteomes in distinct subcellular compartments of astrocytes and neurons in vivo. Here, we describe step-by-step protocols for the in vivo expression of BioID2, followed by biotinylation and subsequent purification of biotinylated proteinsBiotinylationbiotinylated proteins for mass spectrometryMass spectrometry (MS) analysis using a brain-wide adeno-associated viral (AAV) approach.