Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is considered one of the world’s 20 most neglected tropical diseases by the World Health Organisation (WHO). T. cruzi is endemic to 21 countries within the Americas, infects approximately 28 000 people and causes some 12 000 deaths, per year. The drugs currently used to treat T. cruzi infection have questionable efficacy and associated toxicity. Inevitably, the result is ineffective or incomplete treatment, therefore new compounds are needed for the drug discovery pipeline. Attrition of compounds is an ongoing issue for Chagas discovery and improvement of in vitro assays to further understand the efficacy and mode of action of new compounds would support new discoveries. We have developed a high-throughput, high-content assay to assess compound activity against intracellular T. cruzi amastigotes utilising the fluorescent markers Hoechst and HCS CellMask Green. To understand the replicative capability of T. cruzi, we have employed a fluorescent flou-488 marker to identify incorporation of a collection of 6 thymidine analogues into T. cruzi DNA, visualised using click chemistry. Analogues were co-incubated with intracellular T. cruzi parasites over time to identify 5-ethynyl-2′-deoxyuridine (EdU) as the most effectively incorporated into parasite DNA. Identification of DNA synthesis by EdU has previously been used to label replicating mammalian cells. EdU was employed to develop a novel image-based assay to assess the activity of compounds against parasite replication. The effects of the drugs used to treat Chagas disease on T. cruzi replication were investigated, in addition to posaconazole, which causes a sub-efficacious effect against the parasite in vitro and has failed clinical trials to treat Chagas disease. Active compounds against T. cruzi from the Medicines for Malaria Venture (MMV) Pathogen Box were also assessed for their effect upon parasite replication. This methodology provides new insights into the MOA and static/cidal nature of the action of these compounds and drugs and is a promising tool to aid the prioritisation of compounds in drug discovery.