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Chemotactic responses play a significant role during Leishmania (V.) braziliensis differentiation through its life cycle and during infection. The aim of this description has been to portray the modified “two-chamber capillary chemotaxis assay” as a technique useful for quantitative in vitro evaluation of Leishmania chemotaxis after reviewing the methods described until now to assess chemotaxis in vitro in Leishmania sp. This valued simple and reproducible method convenient for parasite migration determination, was tested by the use of controlled changes in monosaccharide (D-glucose and D-fructose) concentrations as referent ligands. The validation of the method demonstrates that this technique is useful to evaluate the relationship existing between parasite migration towards the monosaccharides and sugar concentration. This means that within specific ranges, parasites attracted by the monosaccharide migrate towards more concentrated solutions and accumulate (higher number of parasites) at that spot. Interestingly, both the time course of the experiment and the osmolality of the solution influence parasite migration capacity. Our validation suggests that this improved methodology quantitatively evaluates taxis of Leishmania towards/against different substances. On the basis of our herein presented data, we conclude that this technique is a novel, rapid and reliable screening method to evaluate chemotaxis in Leishmania.•The two-chamber capillary chemotaxis assay was standardized for Leishmania.•The technique is useful to quantitatively evaluate in vitro chemotaxis in Leishmania.•Parasite migration was characterized by monosaccharide chemical gradients.•This assay is a novel, rapid and reliable screening method to evaluate chemotaxis. Contain between 1 and 3 bullet points highlighting the customization rather than the steps of the procedure., Graphical abstract Image, graphical abstract