ackground: Pretreatment of semen samples of the infertile patients and in vitro preparation techniques with the certain sperm stimulant agents prior to use for assisted reproductive techniques (ART) has been reported to have beneficial effects on pregnancy rates. Objectives: The objective of this study is to improve certain sperm function parameters in vitro of infertile patients semen by using novel combination motility stimulants substances that improved medium to be used for ART in future. Patients and Methods: One hundred infertile men involved in the current study. They were divided into four groups. Each semen sample was divided into four portions. One part was considered as a control and in vitro activated by using culture medium only. The other three portions were considered as treated groups and activated in vitro by adding pentoxifylline(PX,1mg/ml) and/or L-carnitine (LC,0.5mg/ml) to the culture media. Certain sperm function parameters were examined before and following in vitro activation using layering and sedimentation techniques. Results: The results showed a highly significant (P<0.001) improvement in the sperm concentration and percentage of progressive sperm motility grade(A), grade (B) and grade (A+B) with a highly significant (P<0.001) increase in the percentage of morphologically normal sperm (MNS) after addition of PX and/or LC to the culture medium in comparison with control medium using layering and sedimentation(layering under paraffin) techniques in the infertile patients. Conclusion: The results of this study indicate that adding 20%PXand/ or LC to the culture medium for in vitro sperm activation using layering and sedimentation techniques leads to an improvement in certain sperm function parameters.