Both biotic and abiotic stress activate the oxylipin pathway in plants. As reactive electrophile species (RES), some oxylipins are expected to bind cellular nucleophiles in a Michaël-type addition reaction. Using the HPLC-tandem mass spectrometry techniques, we have established the analytical basis for the investigation of oxylipin conjugation to glutathione (GSH) in plant extracts. The GSH adducts to the four keto fatty acid isomers issued from both linoleic and linolenic acids were first produced and their mass spectrometric features analyzed in the positive electrospray ionization mode. In all cases, the main fragmentation (MS2 mode) of the pseudomolecular ion leads to the neutral loss of a glutamyl moiety (-129 Da), affording an ion that gives structural information upon an additional fragmentation (MS3 mode). The glutamyl loss was confirmed by the analysis of other GSH adducts to oxylipin RES and appeared as being characteristic of GSH adducts. It is thus proposed to search GSH adducts in plant extracts by HPLC-MS/MS, using initially the neutral loss mode and then the MS2 mode to further characterize the identified compounds. This methodology was successfully applied to the analysis of GSH adducts upon infiltration into leaves of the four previous keto fatty acids at 5 mM, a concentration inducing cell death. The production of GSH adducts to oxylipin RES was observed for the first time in plant tissues. Furthermore, the levels of adduct production explain in part the observed GSH depletion. These results support the role of RES in altering protein activities and cellular redox balance of plant cells, via addition reactions to cellular nucleophiles.