Eicosanoids constitute a diverse class of bioactive lipid mediators that are produced from arachidonic acid and play critical roles in cell signaling and inflammatory aspects of numerous diseases. We have previously quantified eicosanoid metabolite production in RAW264.7 macrophage cells in response to Toll-like receptor 4 signaling and analyzed the levels of transcripts coding for the enzymes involved in the eicosanoid metabolite biosynthetic pathways. We now report the quantification of changes in protein levels under similar experimental conditions in RAW264.7 macrophages by multiple reaction monitoring mass spectrometry, an accurate targeted protein quantification method. The data complete the first fully integrated genomic, proteomic, and metabolomic analysis of the eicosanoid biochemical pathway.