Level of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) in biological fluids has been widely used as a biomarker to assess oxidative lesions to DNA (1). Commercially available ELISA tests could provide a fast and simple method to measure 8-oxodGuo in human serum although the overestimations attributed to a lack of specificity of antibodies has been frequently observed (2). Interestingly, there are little reports of mass spectrometry techniques attempting to measure 8-oxodGuo in serum. Herein we develop an isotope-dilution LC-MS/MS method coupled with an on-line solid-phase extraction (on-line SPE) to measure serum 8-oxodGuo. Serum sample of 200μl was firstly added 15N5-8-oxodGuo as an internal standard, followed by the addition of acetonitrile (ACN) to remove the proteins from serum. After centrifugation, the supernatant was collected, dried and re-dissolved in 5%MeOH/0.1% FA for on-line SPE LC-MS/MS analysis. This method allows for fast and high-throughput analysis of serum 8-oxodGuo because it does not use a column-based purification process; it is also relatively inexpensive by using common laboratory reagents. The results showed that the overall mean recovery of 8-oxodGuo in serum was ~97 % by our newly developed on-line SPE LC-MS/MS with protein precipitation method. This on-line SPE LC-MS/MS method is now ready for comparison tests with the commercial ELISA tests using healthy volunteer’s serum samples.