This study aimed to assess the correlation between 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dGuo) in semen and urine, and to compare the analytical methods of the isotope-diluted liquid chromatograph-tandem mass spectrometry (LC-MS/MS) coupled with an on-line Solid-Phase Extraction (SPE) and commercial Enzyme- Linked Immunosorbent Assay (ELISA) used for detecting 8-oxo-dGuo as an oxidative DNA damage marker. Semen and urine samples were simultaneously collected from 85 apparently healthy human subjects. An optimized DNA extraction method was employed to extract DNA from sperm while minimizing oxidation of DNA. All of the biological samples were analyzed by LC-MS/MS and ELISA. All of the biological samples were detected with 8-oxodGuo. ELISA consistently detected two to three times higher 8-oxodGuo levels in urine samples than LC-MS/MS. However, there was no significant correlation between measurements of 8-oxo-dGuo levels in urine and semen. In conclusion, the LC-MS/MS coupled with an SPE was a sensitive method to detect and quantify 8-oxo-dGuo in human sperm and urine. Urinary 8-oxo-dGuo may not be a reliable marker for detecting oxidatively damaged DNA in sperm.