Oxidative stress has been implicated in numerous diseases, including arthritis, atherosclerosis, Alzheimer's disease, cancer, diabetes, hypertension, and inflammation. 8-iso-prostaglandin F2α, a member of the F2 isoprostane family, has been well-accepted as a valuable biomarker for the assessment of oxidative stress.We report the development and validation of an ultra-sensitive LC-MS/MS assay for urinary 8-iso-PGF2α measurements in pediatric population.The assay was linear from 0.024 to 20nmol/l (R2=0.99). Recoveries were above 85% and matrix effects were below 5%. The variability was determined at nmol/l concentration: the intra-day variability (%CV) ranged from 3.9% to 4.5% (n=20); and the inter-day variability ranged from 4.3% to 5.7% (n=20). The accuracy of our laboratory developed test was evaluated with a clinical reference laboratory (n=39), and a correlation coefficient of 0.9257 was observed. Reference interval were established to be <0.5ng/mg creatinine in a group of pediatric population (2months–18years, n=123).The precision of the assay will allow for accurate assessment of oxidative stress, and is acceptable for patient testing, particularly in pediatric population.