Chagas disease (CD), caused by Trypanosoma cruzi, is the main parasitic disease in the Western Hemisphere, with an increasing number of cases, especially in non-endemic regions. The disease is characterized by cardiomegaly and mega viscera, nevertheless, the clinical outcome is hard to predict, underscoring the need for further research into the pathophysiology of CD. Even though most basic and translational research involving CD is performed using in vivo models, in vitro models arise as an ethical, rapidly evolving, and physiologically relevant alternative for CD research. In the present review, we discuss the past and recent in vitro models available to study the host-parasite interface in cardiac and intestinal CD, critically analyzing the possibilities and limitations of state-of-the-art alternatives for the CD host-parasite investigation.