Oxidative stress (OS) plays a major role in the pathogenesis of various diseases in humans. OS is a result of an imbalance between reactive oxygen species (ROS) and the biologically available antioxidants that prevent or repair damage that ROS inflict on the host cells. ROS are naturally generated during normal mitochondrial respiration and by oxidative burst during the immune response. Many factors may influence OS, including genetics, diet, exercise, and exposure to environmental toxicants (e.g., tobacco smoke). A nonenzymatic peroxidation product of arachidonic acid (AA), 8-iso-PGF (8-isoprostane), is a validated biomarker of OS that is present in urine as both glucuronide conjugate and free acid. Previous studies report that the conjugated forms of 8-isoprostane can vary between 30 and 80% of the total 8-isoprostane levels. By hydrolyzing the conjugated forms, it is possible to obtain a total (free + conjugated) measurement of 8-isoprostane in urine samples. Here, we describe a robust, automated, and high-throughput method for measuring total urinary 8-isoprostane using a polymeric weak anion-exchange solid-phase extraction (SPE) and isotope-dilution ultrahigh performance liquid chromatography electrospray ionization-tandem mass spectrometry (UHPLC-MS/MS). This method, using a 96-well plate platform, showed good sensitivity (8.8 pg/mL LOD) and used only 400 μL of the sample volume with a cycle time of 11 min. The inter- and intraday precision, calculated from 20 repeated measurements of two quality control pools, varied from 4 to 10%. Accuracy, calculated from the recovery percentage at three spiking levels, ranged from 92.7 to 106.7%. We modified this method to allow for the exclusive measurement of free 8-isoprostane by removing the hydrolysis step. We measured both free and total 8-isoprostane in urine collected from 30 cigarette smokers (free: 460 ± 78.8 pg/mL; total: 704 ± 108 pg/mL) and 30 nonusers of tobacco products (free: 110 ± 24.2 pg/mL; total: 161 ± 38.7 pg/mL). This method is robust, accurate, and easily adaptable for large population studies.Copyright © 2020 American Chemical Society.