A C T Fatty acid desaturases are key enzymes in lipid metabolism. They introduce double bonds between defined carbons of the fatty acyl chain and catalyze rate-limiting steps in the biosynthesis of polyunsaturated fatty acids. For decades, in vitro desaturase activities have been determined by using radiolabeled fatty acids as substrates, incubated with tissue or cell fractions containing membrane-bound desaturases. However, handling radioactivity is being increasingly complicated due to safety and regulatory concern. Radiolabeled fatty acids are also expensive and many of them are not commercially available. There is therefore a crucial need to develop new methods. Although methods using unlabeled fatty acids as substrates have recently been validated, they are well suited for large tissue samples and did not achieve the same sensitivity as the radioactive ones. Here, we show that negative chemical ionization GC-MS on stable isotope-labeled fatty acids, derivatized to pentafluorobenzyl esters, now offers this opportunity, because of its high sensitivity in the selected ion monitoring mode. By using this simple and affordable improved method, we measured the kinetic parameters of mouse liver Δ6-desaturase for its two main substrates (C18:2 n-6 and C18:3 n-3; 10-13 µM). Moreover, this method enabled to compare Δ5desaturase apparent Km values (19-22 µM) for its two main substrates (C20:3 n-6 and C20:4 n-3). Finally, we reevaluated the controversial effect of freezing on desaturase activities by using both frozen rat tissues and cryopreserved human hepatocytes. This safe, reliable and sensitive method may be applied to other enzymatic activities involving fatty acids (elongation, hydroxylation) in miniaturized samples.