Chagas disease (American trypanosomiasis) is the parasitic disease with the greatest health impact in Latin America and a growing health problem in non-endemic countries due to migratory flows. Complete conversion to negative serology using standard tests can take more than 10 years and is achieved in approximately 30% of treated patients. It is unknown whether the antibodies detected in the circulation of chronically infected subjects derive from the activation of naïve B cells or memory B lymphocytes that continually differentiate into plasmablasts in the phase of parasite persistence or from long-lived plasma cells that persist for a long time in the absence of antigen. Because plasmablasts are rarely present in the circulation in the absence of antigen or of viable replicating pathogens, measurement of T. cruzi-specific plasmablasts after anti-parasite treatment could provide a rapid assessment of treatment efficacy relative to the measurement of T. cruzi-specific antibodies, whose decay can be masked by antigen-independent persistence of antibodies derived from long-lived plasma cells. The enzyme-linked immunospot (ELISPOT) assay is a powerful tool to evaluate antigen-specific immune responses at the single-cell level. Herein, we describe the ELISPOT assay to measure the levels of antibody-secreting B lymphocytes (ASCs) and memory B cells specific for T. cruzi in PBMCs from subjects with chronic Chagas disease.