Various physiological imbalances lead to reactive oxygen species (ROS) overproduction and/or increases in lipoxygenase (LOX) activities, both events ending in lipid peroxidation of polyunsaturated fatty acids (PUFAs). Besides the quantification of such a process, the development of tools is necessary in order to allow the identification of the primary cause of its development and localization. A biochemical method assessing 9 LOX, 13 LOX and ROS-mediated peroxidation of membrane-bound and free PUFAs has been improved. The assay is based on the analysis of hydroxy fatty acids derived from PUFA hydroperoxides by both the straight and chiral phase high-performance liquid chromatography. Besides the upstream products of peroxidation of the 18:2 and 18:3 PUFAs, products coming from the 16:3 were characterized and their steady-state level quantified. Moreover, the observation that the relative amounts of the ROS-mediated peroxidation isomers of 18:3 were constant in leaves allowed us to circumvent the chiral analyses for the discrimination and quantification of 9 LOX, 13 LOX and ROS-mediated processes in routine experiments. The methodology has been successfully applied to decipher lipid peroxidation in Arabidopsis leaves submitted to biotic and abiotic stresses. We provide evidence of the relative timing of enzymatic and non-enzymatic lipid peroxidation processes. The 13 LOX pathway is activated early whatever the nature of the stress, leading to the peroxidation of chloroplast lipids. Under cadmium stress, the 9 LOX pathway added to the 13 LOX one. ROS-mediated peroxidation was mainly driven by light and always appeared as a late process.