A novel analytical approach combining solid-phase microextraction (SPME)/gas chromatography ion trap mass spectrometry (GC-IT-MS) was developed for the detection and quantification N,N-dimethyl- tryptamine (DMT), a powerful psychoactive indole alkaloid present in a variety of South American indigenous beverages, such as ayahuasca and vinho da jurema. These particular plant products, often used within a religious context, are increasingly consumed throughout the world following an expansion of religious groups and the availability of plant material over the Internet and high street shops. The method described in the present study included the use of SPME in headspace mode combined GC-IT-MS and included the optimization of the SPME procedure using multivariate techniques. The method was performed with a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber in headspace mode (70 min at 60 °C) which resulted in good precision (RSDo8.6%) and accuracy values (71–109%). Detection and quantification limits obtained for DMT were 0.78 and 9.5 mg L 1, respectively and good linearity (1.56–300mgL 1, r21⁄4 0.9975) was also observed. In addition, the proposed method showed good robustness and allowed for the minimization of sample manipulation. Five jurema beverage samples were prepared in the laboratory in order to study the impact of temperature, pH and ethanol on the ability to extract DMT into solution. The developed method was then applied to the analysis of twelve real ayahuasca and vinho da jurema samples, obtained from Brazilian religious groups, which revealed DMT concentration levels between 0.10 and 1.81 g L .