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Lipopolysaccharide (LPS), a glycolipid component of the outer membrane of Gram-negative bacteria, is a potent initiator of the macrophage’s innate immune response. LPS triggers downstream signaling by selectively recruiting and activating proteins in cholesterol-rich membrane microdomains called lipid rafts. We applied proteomic analysis to macrophage detergent-resistant membranes (DRMs) during an LPS exposure time course in an effort to identify and validate novel events occurring in macrophage rafts. Following metabolic incorporation in cell culture of heavy isotopes of amino acids Arginine and Lysine (Arg-13C6, Lys-13C6) or their light counterparts, a SILAC (Stable Isotope Labeling of Amino acids in cell Culture)-based quantitative, liquid chromatography-tandem mass spectrometry proteomics approach was used to profile LPS-induced changes in the lipid raft proteome of RAW 264.7 macrophages. Unsupervised network analysis of the proteomics dataset revealed a marked representation of the ubiquitin-proteasome system, as well as changes in proteasome subunit composition following LPS challenge. Functional analysis of DRMs confirmed that LPS causes selective activation of the proteasome in macrophage rafts and proteasome inactivation outside of rafts. Given previous reports of an essential role for proteasomal degradation of IκK