Oxylipins, derived from polyunsaturated fatty acids, are essential in inflammation, immuneresponse, and vascular function. They are valuable as potential biomarkers for detecting healthchanges. Given the physiological similarities between dogs and humans, studying oxylipins indogs can model human diseases. During pregnancy, the maternal metabolome affects newbornoutcomes, and identifying new biomarkers for early and non-invasive pregnancy diagnosis indogs is vital due to the unique inflammatory environment. This approach improves dogbreeding and welfare. Since oxylipin isolation from dog urine has yet to be studied, this researchintroduces an optimized method using solid-phase extraction (SPE) with liquid chromatographyand tandem mass spectrometry (LC-MS/MS). The study finds the reversed phase sorbentutilized by the Bond Elut C18 cartridge to be suitable for the extraction of urinary oxylipins.The calibration curve, ranging from 0-60 ng/mL, exhibited good linearity (R² > 0.99) for mostanalytes, with limits of detection (LOD) ranging from 0.1 to 3.9 ng/mL, showcasing highsensitivity. This study used the design of experiments (DOE) method to find optimal conditionsto separate oxylipins by C18 SPE. Analysis of the model resulted in high R², Q², model validity,and reproducibility values, which affirmed the robustness, validity, and ability for predictions.The model showed that methanol composition in the washing stage, washing and elutionvolume, and elution solvent are critical factors, individually or interactively, influencing therecovery and matrix effects. Interactive factors analyses revealed intricate dependencies amongvariables. The optimal SPE conditions were at 4 mL washing solution of 20% methanol in waterand 2.5 mL methanol for elution of selected oxylipins. The mixed mode SPE was also examinedat different pH values to find a suitable separation mechanism for isolating oxylipins in dogurine samples, which showed the suitability of reversed-phase mechanism on oxylipinseparations. Overall, this study systematically investigated the separation of oxylipins usingvarious SPE sorbents, providing a solid foundation for future studies on the characterization ofoxylipins in dog urine samples.