Macrophages produce various kinds of lipid mediators including eicosanoids and platelet-activating factor. Since they are produced from common precursors, arachidonic acid-containing phospholipids, regulations of metabolic pathways underlie the patterning of lipid mediator production. Here, we report a pathway-oriented profiling strategy of lipid mediators by a newly developed multiplex quantification system. We profiled mouse peritoneal macrophages in different activation states. The analysis of kinetics revealed the differences in the production time course of various lipid mediators, which also differed by the macrophage types. Scatterplot matrix analysis of the inhibitor study revealed correlations of lipid mediator species. The changes of these correlations provided estimates on the effects of lipopolysaccharide priming. We also found a highly linked production of 11-hydroxyeicosatetraenoic acid and prostaglandin E2, implying the in vivo property of cyclooxygenase-mediated 11-hydroxyeicosatetraenoic acid production. The present approach will serve as a strategy for understanding the regulatory mechanism of lipid mediator production.