The protozoan parasite, Trichomonas vaginalis (Tv) causes trichomoniasis, the most common, non-viral, sexually transmitted infection in the world. Only two closely related drugs are approved for its treatment. The accelerating emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health. There is an urgent need for novel effective anti-parasitic compounds. The proteasome is a critical enzyme for T. vaginalis survival and was validated as a drug target to treat trichomoniasis. However, to develop potent inhibitors of the T. vaginalis proteasome, it is essential that we understand which subunits should be targeted. Previously, we identified two fluorogenic substrates that were cleaved by T. vaginalis proteasome, however after isolating the enzyme complex and performing an in-depth substrate specificity study, we have now designed three fluorogenic reporter substrates that are each specific for one catalytic subunit. We screened a library of peptide epoxyketone inhibitors against the live parasite and evaluated which subunits are targeted by the top hits. Together we show that targeting of the β5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of β5 plus either β1 or β2 results in improved potency.