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, 200µL of supernatant was placed into an autosampler vial for LC-MS/MS analysis. The lower limit of quantitation (LLOQ) was 3pg/mg. The resulting calibration curve for dimethyltryptamine fit the expression y=281.50213x+0.00231 (R2=0.992). Acceptable intraday and interday precision (RSD <15%) and accuracy (92%-113%) were achieved. The dilution integrity was deemed acceptable based on accuracy (96%) and precision (1.8%). The validated method was successfully applied to 28 forensic cases. The concentrations of dimethyltryptamine ranged from 3 to 1109pg/mg.