Primary screening of possible antileishmanial compounds is currently based either on the use of promastigotes, which are not the clinically relevant stage, or on the time-consuming intracellular amastigote test. With the aim of discovering new antileishmanial derivatives, a semi-automatized method using Leishmania axenic amastigotes and a fluorometric growth indicator (Alamar Blue) was developed. This assay was evaluated in a comparative study of the susceptibility of promastigotes, axenic amastigotes and intracellular amastigotes of Leishmania mexicana with standard drugs used in antileishmanial therapy (meglumine antimoniate, amphotericin B, ketoconazole and allopurinol) and recently developed ether lipid compounds (edelfosine and miltefosine). The results showed that while inhibitory concentration 50s (IC50s) of axenic and intracellular amastigotes were similar for all of the drugs tested, promastigotes and intracellular amastigotes IC50s differed for three of the six compounds. In conclusion, this axenic amastigote model allowed us to combine the ease of in vitro drug screening on promastigote with the reliability of the IC50 determination on intracellular amastigotes.