Ayahuasca is a psychoactive beverage widely used in religious ceremonies in Amazonia. Dimethyltryptamine is the main active compound of ayahuasca. Dimethyltryptamine has many hazardous effects, including hallucinations. In the present study, a fast and reliable UPLC-MS/MS method was developed and validated for the quantitation of dimethyltryptamine in hair samples. Twenty-milligram hair samples were pulverized with methanol below 4 °C. After ultrasonication, centrifugation and filtration, 200 μL of supernatant was placed into an autosampler vial for LC-MS/MS analysis. The lower limit of quantitation (LLOQ) was 3 pg/mg. The resulting calibration curve for dimethyltryptamine fit the expression y = 281.50213x + 0.00231 (R2 = 0.992). Acceptable intraday and interday precision (RSD