The expanding research interest in the last two decades on reactive oxygen species (ROS), oxidative stress, and male infertility has led to the development of various techniques for evaluating oxidative DNA damage in human spermatozoa. Measurement of 8-hydroxydeoxyguanosine (8-OHdG) offers a specific and quantitative biomarker on the extent of oxidative DNA damage caused by ROS in human sperm. The close correlations of 8-OHdG level with male fertility, sperm function and routine seminal parameters indicate the potential diagnostic value of this technique in clinical applications. On the other hand, single cell gel electrophoresis (SCGE or comet assay) and terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay have also been demonstrated to be sensitive, and reliable methods for measuring DNA strand breaks in human spermatozoa. As certain technical limitations were inherent in each of these tests, it is believed that a combination of these assays will offer more comprehensive information for a better understanding of oxidative DNA damage and its biological significance in sperm function and male infertility.