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Background: Leishmaniases are a group of diseases caused by Leishmania parasites.  Growing of drug unresponsiveness in leishmaniasis patients necessitates the development of new drugs and accor­dingly a suitable assay is needed for evaluation of any modalities.  The aim of this study was to compare four drug assays methods, agar dilution, broth dilution, cylinder plate and disk diffusion, for evaluation of anti-leishmanial drugs on Leishmania promastigotes, using glucantime as a currently availa­ble drug for treatment of leishmaniasis.  Methods: For broth dilution method, different concentration of glucantime was added to the para­site culture (promastigotes of Leishmania), while in cylinder plate method wells were punched in agar gel and filled with different concentration of drug and zone of inhibition was measured in each well. In disk diffusion method, the parasites were cultivated on the surface of agar; filter paper disks were enriched with various concentration of glucantime and were placed on the surface of agar. In agar dilution method, various concentrations of drug were incorporated onto blood agar and the parasites were cultivated on the surface of the agar. Results: A direct correlation was found between the drug concentration and size of inhibitory zones in cylinder plate and disk diffusion methods. These two drug assays methods provided much better performance in comparison with broth and agar dilution methods. Conclusion: Cylinder plate and disk diffusion methods seem to be acceptable methods for susceptibil­ity testing of anti-leishmanial compounds on Leishmania promastigotes.