Introduction: Cutaneous, muco-cutaneous and visceral leishmaniasis occur due to an infection with the protozoan parasite Leishmania. The current therapeutic options are limited mainly due to extensive toxicity, emerging resistance and variation in efficacy based on species and strain of the Leishmania parasite. There exists a high unmet medical need to identify new chemical starting points for drug discovery to tackle the disease. Areas covered: The authors have highlighted the recent progress, limitations and successes achieved in assay development for leishmaniasis drug discovery. Expert opinion: It is true that sophisticated and robust phenotypic in vitro assays have been developed during the last decade, however limitations and challenges remain with respect to variation in activity reported between different research groups and success in translating in vitro outcomes in vivo. The variability is not only due to strain and species differences but also a lack of well-defined criteria and assay conditions, e.g. culture media, host cell type, assay formats, parasite form used, multiplicity of infection and incubation periods. Thus, there is an urgent need for more physiologically relevant assays that encompass multi-species phenotypic approaches to identify new chemical starting points for leishmaniasis drug discovery.