A wide range of biologically active lipids such as eicosanoids, docosanoids, and other lipid mediators are known for their physiological and pathophysiological effects in almost every organ system. It is essential that the full spectrum of these compounds is comprehensively captured, to fully characterize their biological activity. The analytical determination of these lipid mediators is challenging, however, mainly because of their biological diversity and their physicochemical similarity. LC-MS/MS, in particular in combination with high-resolution instruments, is currently the most powerful tool for analysis of lipid mediators. This chapter describes a comprehensive analytical protocol that allows for simultaneous profiling of over 100 polyunsaturated fatty acid (PUFA)-derived metabolites using a single analytical method from a wide range of biological matrices.