Kallikrein 3, commonly known as prostate specific antigen (PSA), is a serine protease of the human tissue Kallikrein gene family. PSA is synthesized in the ductal and acinar epithelium of the prostate gland and secreted into the seminal plasma in high concentrations (0.5 - 2 g/L). A small portion of PSA “leaks” into the systemic circulation, the levels of which increase significantly (30-fold) from prostate cancer tissue than normal prostate tissue. PSA has become a well established tumor marker that aids the diagnosis, staging, and follow up of prostate cancer. The activity of recombinant human KLK3 is measured by its ability to cleave a colorimetric peptide substrate Suc-Arg-Pro-Tyr-pNa in the assay buffer 50 mM Tris, 1 M NaCl, pH 8.0. The rhKLK3 is diluted to 200 ug/ml in activation buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5, then activated with a final concentration of 1 ug/ml Thermolysin at 37 ℃ for 5min. Adding a final concentration of 10 mM 1,10 Phenanthroline to stop the activation. The activated rhKLK3 is diluted to 50 ug/mL in assay buffer. Loading into a clear well plate 50 µL of 50 ug/mL rhKLK3 and start the reaction by adding 50 µL of 2 mM substrate, with a substrate blank containing 50 µL assay buffer, 50 µL substrate, and no rhKLK3. Then read at 405 nm in kinetic mode for 5 minutes. The specific activity of recombinant human KLK3 is > 400 pmol/min/µg.