Background: Measurement of the success of antitrypanosomal treatment for Chagas disease is difficult, particularly in the chronic phase of the disease, because anti-Trypanosoma cruzi antibodies persist in serum for prolonged periods. We studied the effects of nifurtimox administered by two different treatment regimens on the T. cruzi calcium-binding flagellar protein F29 in children diagnosed with Chagas disease measured using an enzyme-linked immunosorbent assay (ELISA) technique (ELISA F29). Methods and principal findings: In a phase 3, randomized, double-blind, parallel-group, historically controlled study (ClinicalTrials.gov NCT02625974), blood samples obtained from children diagnosed with Chagas disease and treated with nifurtimox for either 60 days or 30 days were analyzed using an ELISA with an F29 recombinant protein as the antigen, as well as conventional serological tests (recombinant ELISA and indirect hemagglutination assay). In an exploratory approach, serological response to nifurtimox treatment was evaluated for 4 years post-treatment. In both treatment groups, the number of patients with negative ELISA F29 values increased over the period of observation. The incidence rate of negative seroconversion using ELISA F29 was 22.94% (95% CI: 19.65%, 26.63%) in the 60-day treatment group and 21.64% (95% CI: 17.21%, 26.86%) in the 30-day treatment group. In the subpopulation of patients who tested seropositive for F29 before nifurtimox treatment, 88 patients (67.7%) in the 60-day regimen and 39 patients (59.1%) in the 30-day regimen were F29 seronegative at 4 years post-treatment. All patients who had a positive ELISA F29 test at baseline and seroconverted to negative measured by conventional serology reached seronegativity in ELISA F29 earlier than in conventional serology. Conclusions: The results demonstrate a serological response to treatment with nifurtimox measured by the ELISA F29 test in children diagnosed with Chagas disease. The F29-based ELISA can be considered a potential early marker of response to antitrypanosomal therapy for Chagas disease. Trial registration: ClinicalTrials.gov NCT02625974. Author summary: Chagas disease (American trypanosomiasis) is a potentially life-threatening parasitic disease caused by Trypanosoma cruzi. Measurement of the success of antitrypanosomal treatment is difficult, particularly in the chronic phase of the disease, because serum anti-T. cruzi antibodies persist for prolonged periods even after complete elimination of the parasite. Thus, markers for early assessment of response to antitrypanosomal therapy could be useful tools to monitor response to drug treatment in Chagas disease. In this prospective study, we analyzed serum samples obtained from children diagnosed with Chagas disease and treated with nifurtimox (an antitrypanosomal drug with proven effectiveness). Before treatment and at each annual visit for the 4 years after treatment, we measured levels of antibodies to a specific antigenic protein (F29) of T. cruzi parasite using an established immunological technique; enzyme-linked immunosorbent assay (ELISA). The number of patients with negative ELISA F29 results increased over the period of observation after the end of treatment. At 4 years post-treatment, we found seronegative ELISA F29 test results in 67.7% of the patients in the 60-day nifurtimox regimen and in 59.1% of the patients in the 30-day nifurtimox regimen. The F29-based ELISA can be considered a useful method to monitor responses to drug treatment in children with asymptomatic Chagas disease. [ABSTRACT FROM AUTHOR]