BACKGROUND: Semen extender is liquid diluents which are added to semen to preserve its fertilizing ability. The addition of extender to semen protects the sperm against possible damage by toxic seminal plasma, as well as providing nutrients. AIM AND OBJECTIVE: The hypotheses of the study are to evaluate, if there is any significant change in the protein concentration in each fractions of human semen before and after freezing with prepared semen extender. Then we are going to correlate the semen parameters with thee fresh semen total protein concentration. RESULTS: After preserving with the extender, all the semen samples shows statistically significant difference (p<0.001) in case of protein concentration. CONCLUSION: There is a significant different (p<0.001) in protein concentration in various fractions before and after freezing. Also the prepared semen extender can be used in Assisted Reproductive centres (ART). © CryoLetters.