Abstract Background Urine 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is a specific biomarker of oxidative stress. We evaluated a modified LC–MS/MS assay for urine 8-oxodG and determined biological variation in healthy adults. Method Untreated urine was injected into an isocratic LC–MS/MS system (positive-ion MRM mode). Urine 8-oxodG in 51 healthy volunteers was measured; within- and between-day variations in 23 healthy volunteers were investigated. Results Dose–response was linear to 452 nmol/l; limit of detection = 2.3 nmol/l; within-run and between-run CVs were < 3.0% and < 4.7%, respectively; recovery = 97%–101%; accuracy = 97.7–103.5%. Urine 8-oxodG (median, mean [SD]): 1.70, 1.70[0.60]nmol/mmol creatinine ( n = 51). Men had higher ( p = 0.027) concentrations than women matched for age and body mass index: mean [SD]: 1.90[1.60]; n = 26 vs. 1.50[0.55]; n = 25. Within- and between-day variations were wide but random. No significant differences were seen overall across time-points within 1 day or at the same time-point across 5 consecutive days. Conclusions The method has advantages of speed and relative simplicity as it does not require sample pre-treatment for 8-oxodG extraction, the use of internal standard or gradient LC elution and has high linearity, specificity, precision and recovery. Biological variation in urine 8-oxodG is wide, but no within- or between-day differences at the group concentration were seen in healthy adults.