Marine diatoms negatively affect reproduction and later larval development of dominant zooplankton grazers such as copepods, thereby lowering the recruitment of the next generations of these small crustaceans that are a major food source for larval fish species. The phenomenon has been explained in terms of chemical defense due to grazer-induced synthesis of oxylipins, lipoxygenase-derived oxygenated fatty acid derivatives. Since this first report, studies about diatom oxylipins have multiplied and broadened toward other aspects concerning bloom dynamics, cell growth, and cell differentiation. Diatom oxylipins embrace a number of diverse structures that are recognized as chemical signals in ecological and physiological processes in many other organisms. In diatoms, the most studied examples include polyunsaturated aldehydes (PUAs) and nonvolatile oxylipins (NVOs). The purpose of this chapter is to provide the analytical tools to deal with identification, analysis and biosynthesis of these compounds. Emphasis is given to identification of the enzymatic steps and characterization of the species-specific lipoxygenases even in absence of the availability of molecular information.