Ayahuasca is a beverage obtained from Banisteriopsis caapi plus Psychotria viridis. B. caapi contains the β‐carbolines harmine, harmaline, and tetrahydroharmine that are monoamine oxidase inhibitors and P. viridis contains N,N‐dimethyltryptamine (DMT) that is responsible for the visionary e!ects of the beverage. Ayahuasca use is becoming a global phenomenon, and the recreational use of DMT and similar alkaloids has also increased in recent years; such uncontrolled use can lead to severe intoxications. In this investigation, liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to study the kinetics of alkaloids over a 24 h period in saliva and serum of 14 volunteers who consumed ayahuasca twice a month in a religious context. We compared the area under the curve (AUC), maximum concentration (Cmax), time to reach Cmax (Tmax), mean residence time (MRT), and half‐life (t1/2), as well as the serum/saliva ratios of these parameters. DMT and β‐carboline concentrations (Cmax) and AUC were higher in saliva than in serum and the MRT was 1.5–3.0 times higher in serum. A generalized estimation equations (GEEs) model suggested that serum concentrations could be predicted by saliva concentrations, despite large individual variability in the saliva and serum alkaloid concentrations. The possibility of using saliva as a biological matrix to detect DMT, βcarbolines, and their derivatives is very interesting because it allows fast noninvasive sample collection and could be useful for detecting similar alkaloids used recreationally that have considerable potential for intoxication.