Astrocytes are increasingly recognized as crucial contributors to neuronal function at synapses, axons, and somas. Reliable methods that can provide insight into the astrocyte proteins at the neuron-astrocyte functional interface are highly desirable. Here we conducted a mass spectrometry analysis of Percoll® gradient isolated gliosomes, a viable preparation of glial subcellular particles often used to study mechanisms of astrocytic transmitter uptake and release and their regulation. Gliosomes were compared with synaptosomes, a preparation containing the neurotransmitter release machinery, and accordingly synaptosomes were enriched for proteins involved in synaptic vesicle mediated transport. Interestingly, gliosome preparations were found to be enriched for different classes of known astrocyte proteins, such as VAMP3 (involved in astrocyte exocytosis), Ezrin (perisynaptic astrocyte cytoskeletal protein), and Basigin (astrocyte membrane glycoprotein), as well as for G-protein mediated signaling proteins. Together, these data provide the first detailed description of the gliosome proteome and show that gliosomes can be a useful preparation to study glial membrane proteins and associated processes.