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The therapeutic armentarium against leishmaniasis is very restricted, using mainly up to now drugs showing several side effects and resistance. Consequently, there is a real need to find new compounds as alternatives for the treatment of leishmaniasis. However, most of the classic antileishmanial primary screening assays are not suitable for measuring the cytotoxicity of large number of drug candidates because of the manipulations required. We have established a new assay that incorporate a fluorometric growth indicator based on the detection of a metabolic activity in culture medium after the chemical reduction of alamar Blue® by cells. This antileishmanial test was evaluated on amphotericin B, meglumine antimoniate, allopurinol, ketoconazole and edelfosine. The results reported show the advantages of this fluorochrome on the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for Leishmania cytotoxicity measurement. Moreover, this study is the first report of the use of alamar Blue® fluorogenic assay for activity assessment of potential antileishmanial drugs against promastigotes.