Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad-active degradome, and the immunoproteome were obtained by using 2-DE, 2-D-zymograms, 2-D-Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty-nine silver-stained spots were detected in the region of 200-21 kDa of parasite protease-resistant extracts. A similar proteolytic pattern was observed in the 2-D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4-like, TvCP12, TvCPT, TvLEGU-1, and another legumain-like CP). The major reactive spots to T. vaginalis-positive patient sera by 2-D-WB corresponded to four papain-like (TvCP2, TvCP4, TvCP4-like, TvCPT), and one legumain-like (TvLEGU-1) CPs. The genes of TvCP4, TvCPT, and TvLEGU-1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture-positive patient sera in 1-D-WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.