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Chagas disease, caused by the protozoan intracellular parasite Trypanosoma cruzi, is a highly neglected tropical disease, causing significant morbidity and mortality in central and south America. Current treatments are inadequate, and recent clinical trials of drugs inhibiting CYP51 have failed, exposing a lack of understanding of how to translate laboratory findings to the clinic. Following these failures many new model systems have been developed, both in vitro and in vivo, that provide improved understanding of the causes for clinical trial failures. Amongst these are in vitro rate-of-kill (RoK) assays that reveal how fast compounds kill intracellular parasites. Such assays have shown clear distinctions between the compounds that failed in clinical trials and the standard of care. However, the published RoK assays have some key drawbacks, including low time-resolution and inability to track the same cell population over time. Here, we present a new, live-imaging RoK assay for intracellular T. cruzi that overcomes these issues. We show that the assay is highly reproducible and report high time-resolution RoK data for key clinical compounds as well as new chemical entities. The data generated by this assay allow fast acting compounds to be prioritised for progression, the fate of individual parasites to be tracked, shifts of mode-of-action within series to be monitored, better PKPD modelling and selection of suitable partners for combination therapy. Author summary: Chagas disease is caused by the single cell protozoan parasite Trypanosoma cruzi. Millions of people suffer from this disease in central and south America, which frequently causes heart disease and can result in death. Chagas disease is classified as a neglected tropical disease due to the lack of investment in development of new medicines. The currently available medicines are inadequate as they require long treatments, often with severe side-effects. To develop new medicines, it is critical to build laboratory assays and tools that help predict the ability of new compounds to cure patients. Rate-of-kill assays measure how quickly compounds can kill parasites, providing a route to differentiate promising compounds from poor ones. Here, we describe development of an advanced rate-of-kill assay that, unlike existing assays, can monitor the same cell population over the duration of compound treatment. Using live-cell microscopy, parasite-infected host cells and their response to compound treatment can be continuously monitored. This enables better defined rate-of-kill profiles to be produced, in turn allowing better informed decisions on subsequent compound progression. Here, we report the live-imaging rate-of-kill profiles for several key compounds, including current drugs and compounds in clinical development. [ABSTRACT FROM AUTHOR]