Trichomonas vaginalis causes trichomonosis, the most common, non-viral sexually transmitted disease. To test anti-Trichomonas agents, usually many with low water solubility, organic solvents and surfactant agents should be used. Therefore, the minimal inhibitory concentration (MIC) of acetone, methanol, ethanol, isopropanol, DMSO, Tween 20, Tween 80, and Triton X-100 was determined against T. vaginalis isolates using the quantitative resazurin method. Our results showed that solvents and surfactant agents can be employed as vehicles to test bioactive compounds at lower concentrations than MIC values and we suggest acetone and DMSO as preferential. Moreover, a new methodology is established to substitute or to complement the counting of viable trophozoites. The amount of resazurin reduced by T. vaginalis can be quantified by fluorescence spectroscopy, making the test a quantitative determination of cell viability. These results contribute for pharmacological investigations of bioactive compounds that need the use of solvents as solubilization vehicles to test anti-Trichomonas activity.