Trypanosoma cruzi is the causative agent of Chagas’ disease, endemic in 21 countries of Latin America. Migration and travel have distributed Chagas’ disease to other continents. In the absence of a vaccine, there is a high need for new antichagasic drugs. However, the latest clinical trials have yielded disappointing results as it became apparent that posaconazole, a highly potent compound in standard in vitro tests, was unable to cure chronic T. cruzi infections. Meanwhile the failure of posaconazole and other azoles to deliver sterile cidality has been reproduced in refined in vitro test systems that demonstrated surviving forms after posaconazole treatment. The nature of those forms and whether they play a role in vivo has been elusive. Here we propose to generate a T. cruzi reporter strain that expresses fluorescent proteins for in vitro monitoring, or luminescent proteins for in vivo imaging, in a stage-specific way, i.e. different colors in the replicating amastigote and the non-replicating trypomastigote forms of the mammalian host. Such a strain will allow to study stage-specificity of drug action and to better assess monotherapies and combinations for their potential to deliver sterile cure. It will also permit new insights into differentiation of T. cruzi in vivo and will allow to better understand phenomena of co-infection, which will be addressed here using previous Schistosoma mansoni infection in a T. cruzi mouse model. Thus the proposed research will give new insights into the biology of T. cruzi that directly translate into drug discovery tools of improved predictability for therapeutic success.