Background: Seminal oxidative stress (OS) is known as one of the important factors of male infertility through pathogenesis of sperm dysfunction and DNA damage. OS results from variation in reactive oxygen species (ROS) production and ROS scavenging by seminal antioxidants. Objective: The aim of this study was to detect the ROS level by Enzyme-Linked Sorbent Assay (ELISA) in seminal plasma of infertile men before and after activation using the Density Gradient Centrifugation (DGC) and glutathione combined with DGC method. Patients and Methods: This study involved 60 males; the recruited individuals were divided into 3 groups, (20 asthenozoospermic, 20 oligozoospermic and 20 normozoospermic subjects) during the period of attendance to the infertility clinic at High Institute for Infertility Diagnosis and Assisted Reproductive Technologies, Al-Nahrain University. The collected semen samples were obtained, and seminal fluid analysis was assessed. Semen samples were divided into 3 parts. The first part prepared was in vitro sperm characterization before activation, the second part using Density Gradient Centrifugation (DGC) technique, while the last part was prepared using DGC combined with glutathione. Results: Reactive oxygen species concentration showed a significant decrease in the 3 groups of normozoospermia, asthenozoospermia, and oligozoospermia when using the DGC technique with glutathione, compared to both before sperm activation and DGC technique. It is important to refer that for the oligozoospermia the level of ROS significantly decreased only between the DGC and DGC with glutathione. Conclusion: Reactive Oxygen Species (ROS) in semen plasma of infertile men was significantly decreased after activation using DGC with glutathione.