Background: Sperm cells from asthenozoospermia have humble motility, Glutathione is added in the medium to increase sperm motility and improve morphologically normal sperm. Cryopreservation of human spermatozoa is one of the best active and acceptable methods to preserve male fertility. However, cryopreservation may lead to changes in sperm structure and decline in sperm functional parameters. Objective: This study has been designed to investigate the effect of Glutathione on certain sperm functional parameters in Asthenozoospemic men before and after cryopreservation. Materials and Methods:The study included forty five asthenozoospermic infertile men. Ejaculated semen were obtained from patients and divided in to two equal portions, first portion was activated by free FertiCult Flushing medium as (control), second portion activated by Glutathione(GSH) in 5 mM concentration. The samples were cryopreserved in liquid nitrogen for one month. All the samples examined for sperm motility, morphology and vitality before and after cryopreservation-thawing, and comparison in these parameters between the two groups done. Results:In vitro activation by GSH resulted in significant (P<0.05) decrease in sperm concentration, highly significant increase (P<0.001) in sperm motility, highly significant decrease in immotile sperm (grade D) and significant increase (P<0.05) in morphologically normal sperm(MNS) compared with pre activation. After cryopreservationthawing, there was significant difference (P<0.05) in certain sperm functional parameters compared with control. Conclusion: There is positive effect and improvement in certain sperm functional parameters after in vitro sperm activation by GSH supplemented medium, before and after cryopreservation.