Trypanosoma cruzi, the aetiological agent of Chagas disease, is a genuine parasite with a tremendous genetic diversity and a complex life cycle, causing complicated pathogenesis. The treatment of the disease has been studied by scientists for more than 100 years, but at present Chagas disease is a life-threatening infection and a global public health problem that has no effective treatment and affects 6-8 million people worldwide. Hence, there is an urgent need for effective new drugs to tackle Chagas disease. Here, we describe a comprehensive strategy and a complete in vitro and in vivo phenotypic-based screening in early drug discovery pipeline for the identification of new effective agents against T. cruzi. In short, 22 aza-scorpiand macrocycles were screened in vitro against different T. cruzi strains (including a BZN-resistant strain), and lead compounds were evaluated in vivo after oral administration in both the acute and chronic infections in mouse model. The mode of action was also evaluated at the energetic level.