A new, simple, and original method is described for specific measurement of polyunsaturated fatty acid content in olive oil. This analytical system uses coupled enzymes, lipase and lipoxygenase. The system consists of lipase-catalyzed hydrolysis of triacylglycerol and subsequent lipoxygenation of liberated polyunsaturated fatty acids. The hydroperoxy-fatty acids formed were easily monitored by spectrophotometry at 234 nm. After being optimized, the method was validated in terms of linearity, precision sensitivity, and recovery. Linear calibration graph was obtained in the range 50-500 microg mL(-1), with a correlation coefficient higher than 0.921 and a detection limit (S/N = 3) of 15 microg mL(-1). The precision of the method (relative standard deviation) for within and between days is better than 7% and 12%, respectively. The proposed method was successfully applied to the estimation of polyunsaturated fatty acids level in olive oil samples and results obtained were in excellent agreement with those obtained by the classical official method. The proposed method is accurate, simple, cheap, and can be satisfactorily used for routine analysis of edible oils.