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Timely diagnosis of vertical Trypanosoma cruziinfections involves microscopy-based detection of circulating parasites from peripheral blood, which lacks sensitivity and is operator dependent. Consequently, most children born to T. cruzi–infected mothers are required to undergo serological testing after 9 months, which risks loss to follow-up. Alternatively, the loop-mediated isothermal amplification (LAMP) test for T. cruziDNA offers high analytical and clinical performance and is easy to use in low-complexity laboratories. Recently, we optimized this technique using an ultrarapid DNA extraction method combined with the LAMP in dried blood spots (DBSs) on Flinders Technology Associates cards. The procedure has been implemented in 10 public maternities across Paraguay, Bolivia, and Argentina, involving the training of 14 technicians. Operators' performance was evaluated using a standardized DBS testing panel for harmonization, including negative controls and DBS samples artificially contaminated with T. cruziat 50 and 20 cells/mL. There was strong agreement (ĸ = 0.924) for controls and 50 cells/mL samples, and good agreement (ĸ = 0.718) across all testing panels, even at the detection limit of the test. A prospective study collected 306 DBSs from 222 newborns at birth and/or 2 months, detecting T. cruzimicroscopically in four cases. LAMP identified eight positive cases and perfectly aligned with real-time PCR (ĸ = 1), demonstrating higher sensitivity than microscopic observation for early detection of infection in infants.