Introduction: Ayahuasca is a beverage composed by a mixture of herbs which contain the compound N,N-dimethyltriptamine (DMT) and the β-carbolines. Although its use is legalized in Brazil only for religious and spiritual ceremonies, there is a growing black market specialized in the distribution of these compounds in form of herbal material through internet and mail. The purpose of this work was the development of an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the determination of ayahuasca alkaloids and its application in seized ayahuasca products. Methods: An aliquot of seized products was weighted and diluted with methanol. An aliquot of this solution was added with internal standard (DMT-d6), followed by injection in the analytical system. Results: The limit of quantitation was 10 ng/mL for DMT and 25 ng/mL for harmine, harmaline and tetrahydroharmine. The concentration ranges used were 10–100 ng/mL for DMT, harmine and harmaline and all analytes presented a coefficient of determination (r2) ≥ 0,99. Analysis of four seized samples presented concentrations of DMT ranging between 31.5 and 46.5 mg/g. Presence of β-carbolines was not detected in the products. The variability of DMT concentrations can be correlated with the potential intoxications described in the literature. Conclusion: This work successfully established a determination method for ayahuasca alkaloids in herbal material. In addition, the workflow proved to be simple, rapid and useful to estimate the concentration of psychoactive compounds in seized materials, leading to further investigation of ayahuasca ritualistic or recreational exposure. © 2020 Elsevier B.V.