This chapter outlines the standardization of serodiagnostic protocols for Trypanosoma cruzi, with an emphasis on optimizing enzyme-linked immunosorbent assays (ELISAs). The standardization process aims to enhance the reliability and reproducibility of diagnostic outcomes across diverse epidemiological settings. The methodology includes detailed descriptions of materials, buffers, and reagents, as well as protocols for antigen coating, assay optimization, and troubleshooting. Performance parameters such as sensitivity, specificity, and predictive values are evaluated using robust statistical analyses to ensure diagnostic accuracy. This comprehensive approach facilitates the validation of diagnostic tools critical for effective disease management and public health strategies.