Visceral leishmaniasis is a neglected tropical disease with significant health impact. Current treatments are poor and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation as the disease-causing stage of the parasite resides inside human host cells. Assessing drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. Here we present a 384 well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems as well as a human counterscreen cell line, and shows that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened in both systems. This resulted in the identification of seven new anti-leishmanial compounds and revealed a high false positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of the axenic assay and discuss how modifications to the axenic assay may render it more suitable for hit-discovery.