The loss of redox homeostasis induced by hyperglycemia is an early sign and key factor in the development of diabetic retinopathy. Due to the high level of long-chain polyunsaturated fatty acids, diabetic retina is highly susceptible to lipid peroxidation, source of pathophysiological alterations in diabetic retinopathy. Previous studies have shown that pterostilbene, a natural antioxidant polyphenol, is an effective therapy against diabetic retinopathy development, although its protective effects on lipid peroxidation are not well known. Plasma, urine and retinas from diabetic rabbits, control and diabetic rabbits treated daily with pterostilbene were analyzed. Lipid peroxidation was evaluated through the determination of derivatives from arachidonic, adrenic and docosahexaenoic acids by ultra-performance liquid chromatography coupled with tandem mass spectrometry. Diabetes increased lipid peroxidation in retina, plasma and urine samples and pterostilbene treatment restored control values, showing its ability to prevent early and main alterations in the development of diabetic retinopathy. Through our study, we are able to propose the use of a derivative of adrenic acid, 17(RS)-10-epi-SC-Δ15-11-dihomo-IsoF, for the first time, as a suitable biomarker of diabetic retinopathy in plasmas or urine.