OBJECTIVE The aim of this study was to determine whether Acridine Orange (AO) can be used as a routine procedure in a physician's office to detect sperm deoxyribonucleic acid (DNA) fragmentation in patients with varicoceles. METHODS Forty-five patients with a mean age of 30.4 years, who had been diagnosed with varicoceles by physical examination and 30 healthy males with a mean age of 28.3 years and without any signs of varicoceles were included in this study. Doppler Ultrasonography was performed to each individual in addition to physical examination. Semen analyses were performed by the same biologist. Sperm morphology was evaluated according to the World Health Organization (WHO) criteria and Kruger's strict criteria. After routine semen analysis, the samples were fixed separately for further examination with AO staining. RESULTS In routine semen analyses, total sperm count, total motile sperm count, and fast direct forward motile and direct forward motile sperm counts were significantly lower in patients with varicoceles, and the immotile sperm count was significantly higher in patients with varicoceles compared to the control group. Kruger's examination revealed a lower sperm count with normal morphology in patients with varicoceles. After AO staining, the ratio of spermatozoa demonstrating red and green coloration were 6.5±11.0% and 93.5±11.0%, respectively in the varicocele group and 1.0±1.0% and 99.0±1.0%, respectively in the control group (p<0.001). CONCLUSIONS Semen analysis using AO staining can be performed under a clinician's office conditions with a fluorescent microscope without any additional equipment. Further studies are needed to validate the AO staining with more extensively used and well-known methods. Therefore, AO staining can be used as a simple and reliable method that can be performed daily in a physician's office in infertility and andrology clinics.