Subcellular fractionation is a valuable procedure in cell biology to separate and purify various subcellular constituents from one another, i.e., nucleus, cytosol, membranes/organelles, and cytoskeleton. The procedure relies on the use of differential centrifugation of cell and tissue homogenates, but additional purification steps now permit the isolation of inter-organellar membrane contact sites. Here, we outline a protocol tailored for the isolation of mitochondria, mitochondria-associated ER membranes (MAMs) and glycosphingolipid-enriched microdomains (GEMs) from the adult mouse brain, primary neurospheres, and murine embryonic fibroblasts (MEFs).