Postsynaptic densities (PSDs) are cytoskeletal specializations present in synaptic contacts in the central nervous system. PSDs have been considered to be subcellular organelles that maintain and cluster the synaptic signal transduction apparatus in direct contact with the actin cytoskeleton and its regulators. Synapses and PSDs are highly dynamic structures that in addition of subserving transmission of information participate in its processing and storage. Therefore, the molecular components of PSDs reveal their signal transduction capacities in health and disease. Here, we present the experimental protocol we have been using for years to isolate PSDs from other cell components, including or excluding detergent-A resistant membranes (or lipid rafts). The protocol can be applied to brain samples of different areas, ages, and mammalian species and is useful to obtain this subcellular organelle in a highly reproducible manner.