In Brazil, orally acquired T. cruzi infection has become the most relevant transmission mech anisms from public health perspective. Around 70% of new Chagas disease cases have been associated with consumption of contaminated food or beverages. Ac¸ai (Euterpe olera cea and Euterpe precatoria) is currently one of the most commercialized Amazonian fruits in the Brazilian and international markets. Therefore, it has become important to incorporate in the production process some procedures to measure out effective hygiene and product quality control required by global market. Molecular methods have been developed for rapid detection and quantification of T. cruzi DNA in several biological samples, including food matrices, for epidemiological investigation of Chagas disease and food quality control. How ever, a high-performance molecular methodology since DNA extraction until detection and quantification of T. cruzi DNA in ac¸ai berry pulp is still needed. Herein, a simple DNA extrac tion methodology was standardized from the supernatant of ac¸ai berry pulp stabilized in a 6M Guanidine-HCl/0.2M EDTA buffer. In addition, a multiplex real time qPCR assay, target ing T. cruzi DNA and an Exogenous Internal Positive Control was developed and validated, using reference from all T. cruzi DTUs and commercial samples of ac¸ai pulp, from an endemic municipality with previous history of oral Chagas disease outbreak. Thus, a high sensitivity qPCR assay, that could detect up to 0.01 parasite equivalents/mL in ac¸ai, was reached. As of the 45 commercial samples analyzed, 9 (20%) were positive for T. cruzi. This high-sensitive, fast, and easy-to-use molecular assay is compatible with most of the labora tories involved in the investigations of oral Chagas disease outbreaks, representing an important tool to the epidemiology, control, and surveillance of Chagas disease.