HPLC with electrochemical detection (HPLC-EC) is a highly sensitive and a selective method for detecting 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker of oxidative DNA damage that is formed from hydroxyl radical attack of guanine residues in DNA. We propose that the noninvasive measurement of oh8dG in urine can be used to estimate in vivo oxidative damage. Application of this assay to urine samples obtained from rats of different ages and various species provide examples of the utility of this assay. The measurement of steady-state levels of oh8dG in DNA combined with the urinary excretion rates of oh8dG and oh8Gua, offer a powerful approach for estimating oxidative DNA damage and its repair. This method will be useful for studies designed to investigate the relationship of oxidative stress in DNA damage and the role of this damage in aging and cancer.