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The parasitic Chagas disease is caused by the protozoan Trypanosoma cruzi, which is mainly transmitted by insect vectors. Other infection routes, both in endemic and in nonendemic areas, include organ and marrow transplantation, congenital transmission, and blood transfusion. Asymptomatic chronic chagasic individuals may have a low and transient parasitemia in peripheral blood and, consequently, they can unknowingly transmit the disease via blood transfusion. Riboflavin and ultraviolet (UV) light pathogen reduction is a method to reduce pathogen transfusion transmission risk based on damage to the pathogen nucleic acids.In this study, we tested the effectiveness of this technology for the elimination of T. cruzi parasites in artificially contaminated whole blood units (WBUs) and thus for decreasing the risk of T. cruzi transfusion transmission. The contaminated WBUs were leukoreduced by filtration and treated with riboflavin and UV light. The level of pathogen reduction was quantified by a real-time polymerase chain reaction (qPCR) and a real-time reverse transcription–polymerase chain reaction (RT-qPCR) as a viability assay.The RNA (cDNA) quantification of the parasites showed a more than 99% reduction of viable T. cruzi parasites after leukoreduction and a complete reduction (100%) after the riboflavin and UV light treatment.Riboflavin and UV light treatment and leukoreduction used in conjunction appears to eliminate significant amounts of viable T. cruzi in whole blood. Both strategies could complement other blood bank measures already implemented to prevent the transmission of T. cruzi via blood transfusion.