N,N-dimethyltryptamine (DMT) is a psychoactive indole alkaloid present in beverages consumed in religious ceremonies and in neo-shamanic rituals all around the world. It is a substance banned in most countries, which makes its acquisition difficult. In Brazil, a beverage rich in DMT named ayahuasca is legally consumed in a religious context. On the other hand, DMT is a controlled drug, enforced by the Brazilian National Health Surveillance Agency (Agência Nacional de Vigilância Sanitária). The present study describes a simple and fast method to obtain N,N-dimethyltryptamine (DMT) from inner barks of Mimosa tenuiflora for the purpose of using it as a chromatographic analytical standard. Fourier transform infrared spectroscopy (FTIR), single and tandem stage mass spectrometry (MS), nuclear magnetic resonance spectroscopy (1H and 13C NMR) and melting point measurements were performed for the structural characterization of N,N-dimethyltryptamine. The results obtained were in agreement with previous literature reports. The purity of the compound (> 95%) was determined using ultraviolet (UV) absorption spectrometry with a tryptamine analytical standard.