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Human biomonitoring of oxidative stress relies on urinary effect biomarkers such as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), and 8-iso-prostaglandin F2α (8-isoprostane); however, their levels reported for similar populations are inconsistent in the scientific literature. One of the reasons is the multitude of analytical methods with varying degrees of selectivity used to quantify these biomarkers. Single-analyte methods are often used, requiring multiple injections that increase both time and cost. We developed a rapid ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method to quantify both urinary biomarkers simultaneously. A reversed-phase column using a gradient consisting of 0.1% acetic acid in water and 0.1% acetic acid in methanol/acetonitrile (70:30) was used for separation. The MS detection was by positive (8-oxodG) and negative (8-isoprostane) ion-mode by multiple reaction monitoring. Very low limit of detection ( 0.999), accuracy (near 100%), and precision (CV < 10%) both for intra-day and inter-day experiments were achieved, as well as high recovery rates (>91%). Matrix effects were observed but were compensated by using internal standards. Our newly developed method is applicable for biomonitoring studies as well as large epidemiological studies investigating the effect of oxidative damage, as it requires only minimal clean up using solid phase extraction.