Objectives: The present study aimed to measure the protamines P1 and P2 concentrations, standard sperm parameters, and also DNA fragmentation and condensation. In addition, the correlations between the above-mentioned variables and the P1/P2 ratio were explored in order to find out whether the ratio could be used as a biomarker for semen quality. Materials and Methods: A total of 272 semen samples were divided into 3 P1/P2 ratio groups: G1 as low (<0. 8), G2 as normal (0. 8-1. 2), and G3 as high (>1. 2) ratios. DNA fragmentation and condensation were evaluated by TUNEL and CMA3 (chromomycinA3) assays and protamine levels (P1 and P2) were measured using acid urea polyacrylamide gel (AU-PAGE) electrophoresis and western blot (WB). Results: In G1, CMA3 positive and DNA fragmentation index (DFI) were negatively correlated (r =-0. 371, P = 0. 001). In addition, P1/P2 ratio had a positive correlation with DFI (r = 0. 652, P = 0. 001); however it had a negative association with CMA3 (r =-0. 623, P = 0. 001). Moreover, G2 demonstrated positive correlations between sperm concentration and P1(r = 0. 257, P = 0. 011) and P2 (r = 0. 277, P = 0. 006), progressive motility, and P1 (r = 0. 352, P = 0. 001). The P2 was positively correlated with progressive motility (r = 0. 380, P < 0. 01). Besides, a positive relationship was found between P1/P2 ratio and CMA3 (r = 0. 333, P = 0. 001). In contrast, normal morphology showed a negative association (P < 0. 05) with P1 (r =-0. 206) and P2 (r =-0. 208). Meanwhile, there was a negative correlation between P1 and DFI (r =-0. 207, P = 0. 041) as well. Finally, in G3, negative correlations were also observed between P1 and normal morphology (r =-. 283, P =. 027) and also P1/P2 ratio and P2 (r =-0. 372, P = 0. 003) while DFI showed a positive correlation with CMA3 (r = 0. 299, P = 0. 019). Conclusions: The results revealed that P1/P2 ratio affected DNA integrity. Therefore, it was found that it could play a crucial role in human sperm quality and function and thus might be used as a predictor of fertility in assisted reproductive technology (ART) treatments.