Background: There are many factors and media affecting sperm characters which are defined by classical parameters namely, concentration, motility and morphology, according to standard World Health Organization (WHO). Objectives: To study the effect of incubation time on certain sperm function parameters following in vitro centrifugation technique by sperm wash, FertiCult medium for the purpose of assisted reproductive techniques (ART) in couples presenting sub-fertility. Methods: Two groups of 40 semen samples (group 1: fertile men with normal semen parameters and group 2: abnormal semen parameters) underwent sperm activation test (SAT) in the ART laboratory, High Institute of Infertility Diagnosis & ART/Al-Nahrain University. Semen analysis was assessed in standard WHO (1999). Wash and spin activation technique by FertiCultTM medium was done for all samples. The tubes which was sealed, inclined vertically and kept at 37°C for 30 minute. The supernatant contained actively motile sperms was transferred to another test tube to separate that active sperm from pellet and record sperm parameters after. Results: There is a significant (p<0.05) improvement in certain sperm parameters after 30min. of incubation following SAT in both groups compared to before activation and following 30min. incubation in SAT. Certain sperm characters are significantly decline in group 2 after 1 hr. incubation compared to before activation. Conclusions: Sperm activation test is very important method to perform before undergone artificial insemination (AIH). Incubation of prepared sperm in vitro by sperm wash, FertiCult medium is benefit for the first half hour to all semen samples to have a success in artificial insemination.