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Ayahuasca, a hallucinogenic beverage used in religious rituals in South America, has become a global phenomenon. Its main active components are the β-carbolines alkaloids, harmine (HRM) and harmaline (HRL), as well as the potent hallucinogen N,N-dimethyltryptamine (DMT). Despite its rising consumption, information regarding possible clinical applications and toxicological effects of ayahuasca is still limited. This study presents the first investigation of the use of sweat for the determination of DMT, HRM and HRL in ayahuasca users during a religious ritual. Sweat is an alternative matrix with advantages over many conventional biological samples, mainly because the collection procedure is non-invasive, easy and simple and samples can be collected without disturbing the religious ritual. In the study, solid-phase extraction was performed under basic conditions. Linearity was observed ranging from 20 to 1500 ng/patch with coefficients of determination (R2) higher than 0.99 for all analytes. The results indicated high selectivity for all investigated analytes, with extraction efficiency exceeding 70%, accuracy ranging from 87.5 to 102.4%, intra-assay precision of 1.85-9.44% and inter-assay precision between 3.34 and 9.85%. The limits of detection were 15 ng/patch for HRM and HRL and 10 ng/patch for DMT. The sweat proved to be a viable option to monitor ayahuasca use., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)