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International audience; Given the growing interest in phytoprostanes (PhytoPs) and phytofurans (PhytoFs) in the fields of plant physiology, biotechnology, and biological function, the present study aims to optimize a method of enzymatic hydrolysis that utilizes bacterial and yeast esterases that allow the appropriate quantification of PhytoPs and PhytoFs. To obtain the highest concentration of PhytoPs and PhytoFs, a response surface methodology/Box−Behnken design was used to optimize the hydrolysis conditions. Based on the information available in the literature on the most critical parameters that influence the activity of esterases, the three variables selected for the study were temperature (°C), time (min), and enzyme concentration (%). The optimal hydrolysis conditions retrieved differed between PhytoPs (21.5°C, 5.7 min, and 0.61 μg of enzyme per reaction) and PhytoFs (20.0°C, 5.0 min, and 2.17 μg of enzyme per reaction) and provided up to 25.1-and 1.7-fold higher contents relative to nonhydrolyzed extracts. The models were validated by comparing theoretical and experimental values for PhytoP and PhytoF yields (1.01 and 1.06 theoretical/experimental rates, respectively). The optimal conditions were evaluated for their relative influence on the yield of individual nonesterified PhytoPs and PhytoFs to define the limitations of the models for obtaining the highest concentration of most considered compounds. In conclusion, the models developed provided valuable alternatives to the currently applied methods using unspecific alkaline hydrolysis to obtain free nonesterified PhytoPs and PhytoFs, which give rise to more specific hydrolysis of PhytoP and PhytoF esters, reducing the degradation of free compounds by classical chemical procedures.