Objective: Currently selection of human sperm for ICSI is based on, motility and morphology but the sperm shape is an inadequate parameter and other procedures should be used for selection of normal sperm. Therefore the aim of this study was to compare the efficiency of Zeta method with Density gradient centrifugation (PurSperm) for separation of sperm with normal chromatin structure Materials and Methods: Semen samples were obtained from 63 patients referred to Isfahan Fertility and Infertility Center. Sperm recovered from zeta method and Density gradient centrifugation (DGC) procedures were evaluated with respect to control (neat semen) group for protamine deficiency, by using Chromomycin A3 (CMA3) staining and for DNA integrity using three different techniques including: sperm chromatin dispersion test (SCD), Acridine orange (AO) ,and TUNEL assay. Results: The results show that the percentage of CMA3 positive sperm have significantly reduced in Zeta and DGC procedures compared to the neat semen . In addition, using three different techniques ,the percentage of DNA damaged sperm have significantly reduced in both procedures compared to the neat semen (control) (p < 0.001)but, the percentage of DNA damaged sperm is significantly lower in the Zeta procedure compared to the DGC procedure with respect to the three tests. The results indicate that the efficiency of Zeta method to separate sperm with normal protamine and intact DNA was higher than DGC procedure Conclusion: It can be concluded that Zeta method is more efficient to recover sperm with minimal DNA damage; however, this method has its own limitations.