Traditional routine semen analysis and biochemical assays are insufficient for the determination of fertility status in individual men. In recent years, clinical evidence has shown that damaged human sperm DNA may adversely affect assisted reproductive outcomes and that the spermatozoa of infertile men possess substantially more DNA damage than the spermatozoa of fertile men. In this study, we combined these two methods to test their clinical significance in assisted reproductive techniques (ART). A total of 302 in vitro fertilization (IVF) and 67 intracytoplasmic sperm injection (ICSI) couples were included in the study. The acridine orange technique (AOT) was used to detect the DNA integrity of the sperm. Correlation analysis showed that the DNA fragmentation index (DFI) was negatively related to the no. of good quality embryos and the clinical pregnancy rate in IVF patients. No significant correlations were found between DFI and ICSI patients. We then combined the results of the routine semen testing of IVF patients with their DFI values. Statistical analysis revealed notable differences in the no. of good quality embryos (P=0.015) and the clinical pregnancy rate (P=0.015) between subgroups divided according to DFI value in a group with normal semen test results (Group 1). In a group with abnormal semen test results (Group 2), the fertilization rate (P=0.034) and the pregnancy rate (P=0.018) showed remarkable variation between DFI subgroups. In conclusion, the detection of damaged DNA in spermatozoa needs to be conducted along with standard semen analysis. This might prove to be a promising predictor of ART outcome, particularly in IVF.