Whereas, monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, using of these proteins has been considered to determination role of surface sperm antigens in fertility mechanism by researchers in field of infertility and reproduction. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. To produce specific clones against human sperm surface proteins were extracted with solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete freund’s adjuvant in the first injection and incomplete adjuvant in the following booster injections. Hybridoma cells, producing ASA, were cloned by limiting dilution. Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with Rat and Mice spermatozoa was examined but they had not any cross reactivity. We are now characterizing the sperm surface antigens recognized by these antibodies, especially aiming at their roles in the process of fertilization.