N,N-dimethyltryptamine (DMT) is a potent hallucinogen found in beverages consumed in religion rituals and neo-shamanic practices over the world. Two of these religions, Santo Daime and União do Vegetal (UDV), are represented in countries including Australia, the United States and several European nations. In some of this countries there have been legal disputes concerning the legalization of ayahuasca con- sumption during religious rituals, a beverage rich in DMT. In Brazil, even children and pregnant women are legally authorized to consume ayahuasca in a religious context. A simple and low-cost method based on matrix solid-phase dispersion (MSPD) and gas chromatography with mass spectrometric detection (GC–MS) has been optimized for the determination of N,N-dimethyltryptamine in Mimosa tenuiflora inner bark. The experimental variables that affect the MSPD method, such as the amounts of solid-phase and herbal sample, solvent nature, eluate volume and NaOH concentration were optimized using an exper- imental design. The method showed good linearity (r = 0.9962) and repeatability (RSD < 7.4%) for DMT compound, with detection limit of 0.12 mg/g. The proposed method was used to analyze 24 samples obtained locally. The results showed that concentrations of the target compound in M. tenuiflora barks, ranged from 1.26 to 9.35 mg/g for these samples.