Background: In developed countries, about (2?4%) of births involve the using of assisted reproductive technologies (ART\'s). In these technologies before using semen samples for insemination firstly it must be processed and specifically sperm preparation methods and materials used to improve sperm functions especially motility in order to ensure increasing in the successful of pregnancy rates next ART\'s. Theophylline pharmacological agents recently commonly used in stimulating motility in human sperm thus by increasing intracellular levels of cyclic adenosine 3?, 5?-monophosphate (cAMP), a molecule involved in formation of sperm energy, which is result of inhibitory properties on phosphdiesterase (PDE) function. Objective: To evaluate whether in vitro sperm activation by Theophylline improves sperm functional parameters as compared with non-activated semen samples. Subjects, Materials and Methods: Sixty male were participated in this study, twenty Normozoospermic subjects and forty Asthenozoospermic patients. Semen samples were collected and seminal fluid analysis was done according to (WHO 1999,2010) guidelines. Each sample was divided equally into three portions, centrifugation was done to remove seminal plasma from all, and then Swim-up technique was dependent for in vitro sperm activation by using FertiCult Flushing medium, Theophylline solution and SpermMobil dilution respectively. Sperm parameters were measured in each activated portion according to (WHO 1999) guidelines. Results: Activation of semen sample groups by using of FertiCult Flushing medium, Theophylline solution and SpermMobil dilution in this study resulted in significant reduction in sperm concentration and highly significant improvement (P<0.001) in the percentage of sperm parameters in comparison with non-activated ones. However, SpermMobil dilution resulted in least reduction in sperm concentration and best result of sperm motility and morphologically normal sperm (MNS). Conclusion: From all comparisons between activation media which have been used in this study with non-activated semen, whatever semen is activated, concentration of sperm will be decreased. Sperm motility and MNS will be improved after activation by FertiCult medium, Theophylline solution and SpermMobil dilution, best results were in increases of sperm motility and MNS which was attributed to SpermMobil dilution.