Growing appreciation for astrocytes as active participants in nervous system development, neurovascular metabolic coupling, and neurological disease progression has stimulated recent investigation into specific astrocyte-secreted proteins that may mediate these functions. The current work utilized SILAC-generated isotope reference proteomes to quantify relative protein abundances between the astrocyte proteome and secretome. Multidimensional GeLC-MS/MS analysis of astrocyte conditioned media and cell lysates resulted in the relative quantification of 516 proteins, 92 of which were greater than 1.5-fold enriched in astrocyte-conditioned media (ACM). Eighty of the ACM-enriched proteins had N-terminal signal peptides, comprising well-known classically secreted proteins, such as apolipoprotein E and SPARC, and several cathepsins that localize to endosomal/lysosomal compartments. The remaining twelve ACM-enriched proteins, such as vimentin, ferritins, and histones, lacked N-terminal signal peptides. Also, 47 proteins contained predicted N-terminal signal peptides but were not enriched in ACM (<1.5-fold), 25 of which were localized to ER, Golgi, or mitochondria membrane-bound compartments. Overall, by combining quantitative proteomics with subcellular localization prediction, an informative description of protein distribution can be obtained, providing insights into protein secretion.