T cells play a pivotal role in the immune response, mediating both direct cytotoxicity and cytokine secretion, as well as shaping the immune environment. In recent years, the activation-induced markers (AIM) assay has emerged as a valuable tool for tracking antigen-specific T cells, enabling a more refined understanding of immune responses in various diseases. By applying this methodology to the context of human chronic Chagas disease (CCD), we aim to better understand the immune dynamics in individuals infected with Trypanosoma cruzi (T. cruzi). Herein, we describe an optimized AIM assay protocol to detect and characterize antigen-specific CD4+ and CD8+ T cells in patients with CCD using different activation markers, such as OX40, CD25, CD40L, CD137, CD69, PD-L1, HLA-DR, and CD38. Thus, this chapter stresses the critical importance of evaluating diverse marker combinations to ensure accurate and reliable results, which will ultimately contribute to advancing both diagnostic and vaccination strategies for T. cruzi infection.