Immunoassay for in vitro quantitative determination of free prostate specific antigen (fPSA) in human serum or plasma. The ratio of fPSA/tPSA can be used for the differential diagnosis of prostate cancer and benign prostatic hyperplasia. Prostate Specific Antigen (PSA) is a member of human prostate kallikrein gene family, it is a serine protease that contains chymotrypsin-like activity.