The ritualistic use of ayahuasca is becoming a global phenomenon. This beverage contains a combination of monoamine oxidase inhibitors (harmine, harmaline, and tetrahydroharmine) and N,N-dimethyltryptamine, the main substance responsible for its visionary effect. The recreational use of similar alkaloids and N,N-dimethyltryptamine has increased in recent years, mainly because of their hallucinogenic effects. In the present study, the concentrations of psychoactive alkaloids in three powder samples seized by the São Paulo State Police and nine ayahuasca aqueous extracts were analyzed by HPLC-DAD in an attempt to distinguish between illicit drugs and the religious beverage. The alkaloids detected (μg/mL) in the ayahuasca aqueous extracts were N,N-dimethyltryptamine (402-2070.3), harmaline (27.5-181.3), harmine (294.5-2893.8), and tetrahydroharmine (849.5-2052.5), whereas, of the three powder samples, one contained only N,N-dimethyltryptamine (82% and 2% w/w, respectively) while the other contained only harmaline (16%, w/w) and harmine (12%, w/w). The ritualistic use of ayahuasca involves oral intake and the probability of overdose is minimized by serotonergic stimulation of vagal pathways, leading to vomiting and diarrhea. In contrast, the recreational use of N,N-dimethyltryptamine involves consumption mainly by smoking or inhalation, both of which markedly increase its bioavailability and the potential for intoxications.