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Publisher Summary This chapter describes the lipoxygenase method that determines the polyunsaturated fatty acids that contain at least one pair of cis-double bonds separated by a methylene group. The methylene group must be located at the ω 8C-atom. The enzyme lipoxygenase catalyzes the oxidation of these pairs of double bonds by atmospheric oxygen. The reaction products are conjugated diene hydroperoxides. The lipoxygenase method is applied in foodstuff chemistry, the oil and fat industry, biochemistry, and clinical chemistry. Typical experimental materials for this method are free fatty acids, fatty acid esters, oils, fats, hydrogenated plant oils, blood plasma and serum, seeds, and micro-organisms. The method allows the determination of as little as 5 μg or 0.018 μmole linoleic acid. The lipoxygenase method is optimum if all the necessary precautions are observed. The chapter discusses the principle, the equipment, and the reagents that are used in this method. It further highlights the preparation of enzyme solutions for that method and the stability of enzyme solutions. All the solutions that contain polyunsaturated fatty acids must be protected against atmospheric oxygen, except during the enzymatic reaction where oxygen is necessary. Storage in oxygen-free conditions is accomplished by displacing the air above the solutions with nitrogen and then stoppering the vessels.