Despite the many studies documenting an increase of sperm DNA damage in subfertile and infertile subjects, determining whether this parameter is relevant for the clinic is still an open question. Indeed, results of clinical investigations on sperm DNA damage and outcome of ART procedures are often conflicting as many factors affect the predictive power of these tests. The techniques used to reveal such damage is one of these factors. Techniques detecting sperm DNA damage are indeed numerous and heterogeneous. In addition, it is not obvious that they reveal the same type of DNA damage and that their results are equivalent. One of the available methods to detect sperm DNA damage is the TUNEL assay. Since it was developed in the 1990s the TUNEL assay has been widely employed, becoming one of the most popular strategies to investigate the origin, the mechanism, and the clinical meaning of sperm DNA damage. Our group has used TUNEL coupled to flow cytometry to investigate sperm DNA fragmentation for about 10 years. According to our experience, this technique presents some pitfalls and limitations in the accuracy and reproducibility of the measures of sperm DNA fragmentation. In this review, we discuss several technical features of TUNEL and report the solutions adopted by our group to overcome some of its limitations.