Background: Sperm cells from infertile patients have poor motility and defected DNA. Pentoxifylline (PX) and Lcarnitine (LC) are added in the medium for in vitro activation to increase sperm motility and improve the pregnancy outcome. However, there are few studies about the effect of PX with LC medium on sperm DNA normality. Objective: The goal of this study is to found out the optimum medium can increase the active motility percentage and reduce sperm DNA damage for asthenozoospermic men using the motility stimulant substances, PX and LC for this purpose. Methods: Semen was collected from 100 infertile men involved in the current study. Each semen sample was divided into four portions. One part was considered as a control group and in vitro activated by using culture medium only. The other portions were considered as treated groups and in vitro activated by adding PX (1mg) and/or LC (0.5mg) to the culture medium. Certain sperm function parameters were examined before and following in vitro activation using layering technique. Sperms DNA damage was detected by using acridine orange (AO) test. Results: The results revealed a highly significant (P<0.01) increment in the mean of sperm concentration (m/ml) percentage of progressive sperm motility grade (A), grade (B) and grade (A+B) and the percentage of morphologically normal sperm (MNS) after using PX and/or LC medium in comparison with control medium .As well as, a highly significant (P<0.001) increase in the percentage of sperm normal DNA by using AO test. Conclusion: It was concluded from the results of the present study that using layering technique with adding PX and/or LC to the culture medium resulting in an improvement in certain sperm function parameters and in the percentage of sperm normal DNA of asthenozoospermic men.