Antigen‐specific T cells are central to the adaptive immune response against T. cruzi infection and underpin the efficacy of on‐going vaccine strategies. In this context, the present study focuses on T‐cell assays that define the parasite‐specificity on the basis of upregulation of TCR stimulation‐induced surface markers. For this purpose, we tested different dual marker combinations (OX40, CD25, CD40L, CD137, CD69, PD‐L1, CD11a, CD49d, HLA‐DR, CD38) to reliably identify activated CD4+ and CD8+ T‐cell populations from PBMCs of chronic Chagas disease (CCD) patients after 12 or 24 h of stimulation with T. cruzi lysate. Results demonstrated that activation‐induced markers (AIM) assays combining the expression of OX40, CD25, CD40L, CD137, CD69 and/or PD‐L1 surface markers are efficient at detecting T. cruzi‐specific CD4+ T cells in CCD patients, in comparison to non‐infected donors, after both stimulation times. For CD8+ T cells, only PD‐L1/OX40 after 24 h of antigen exposure resulted to be useful to track a parasite‐specific response. We also demonstrated that the agnostic activation is mediated by different T. cruzi strains, such as Dm28c, CL Brener or Sylvio. Additionally, we successfully used this approach to identify the phenotype of activated T lymphocytes based on the expression of CD45RA and CCR7. Overall, our results show that different combinations of AIM markers represent an effective and simple tool for the detection of T. cruzi‐specific CD4+ and CD8+ T cells. Activation‐induced markers (AIM) assay is useful to identify T. cruzi‐specific T cells in chronic Chagas disease patients. Several dual AIM markers designate CD4+ T‐cell activation, while only PD‐L1/OX40 identifies parasite‐specific CD8+ T lymphocytes. AIM assay allows the detection of T‐cell agnostic activation mediated by different T. cruzi strains, such as Dm28c, CL Brener or Sylvio.