Background: Intrauterine insemination (IUI) have a low clinical pregnancy rate of 5–13% per cycle. The success of IUI was influenced by the method of preparation and sperm quality. Temperature affects sperm quality. Sperm damage is indicated by the presence of deoxyribonucleic acid (DNA) fragmentation from spermatozoa. Objective: Measuring differences in DNA fragmentation index (DFI) and quality of spermatozoa before and after preparation of the swim-up method at the sperm preparation temperature of 27° and 37°C. Sperm quality measured is concentration, morphology, and progressive motility. Methods: Quasi-experimental laboratory test with pre- and post-test control group design was conducted at the fertility clinic at Dr. Moewardi Hospital. In 20 sperm samples from infertile patients performed the swim-up method at a temperature of 27° and 37°C. Sperm DNA fragmentation was assessed using the chromatin dispersion test (SpermFunc® DNAf). Fragmentation happens if a halo is found <30% of the sperm head volume. Sperm quality assessment based on World Health Organization (WHO) 2010. Data analysis used the Wilcoxon test with a 95% confidence interval and p < 0.05. Results: DNA fragmentation index (DFI) after sperm preparation using the swim-up method at 27°C was lower than 37°C, 17.79 ± 10.88 vs 18.18 ± 12.95, but there was no significant difference (p = 0.765). After sperm preparation using the swim-up method at 27° and 37°C, the sperm concentration was 19.72 ± 13.76 vs 20.55 ± 12.42 (p = 0.512), normal morphology 11.25 ± 5.15 vs 11.6 ± 5.34 (p = 0.626), and progressive motility 82.25 ± 12.77 vs 82.55 ± 11.69 (p = 0.968). Sperm preparation at 27°C has lower DFI, lower sperm quality but without any significant differences. Conclusion: The swim-up method of sperm preparation can be done at room temperature 27°C or 37°C without any significant differences in the DFI and sperm quality.