Psychoactive drugs are compounds that alter the function of the central nervous system, resulting in changes in perception, mood, cognition, and behavior. A subclass of psychoactive drugs, psychedelics, are hallucinogenic drugs that can trigger psychedelic experiences and possible changes in mental perception. The potential use of psychedelics as a therapeutic has led to an increase in clinical research focusing on the treatment of mental disorders including anxiety and depression. There are numerous species belonging to Psychotria and Banisteriopsis which have been reported to contain psychedelic and psychoactive compounds; however, there is a lack of validated analytical methods for raw plant material, which is crucial if these plants are to be commercially cultivated for medicines. This study provides a fully validated method using ultra-high performance liquid chromatography (UHPLC) coupled to mass spectrometry (MS) for the following six compounds: tryptamine, N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), tetrahydroharmine (THH), harmaline, and harmine. The validated method was used to determine the psychoactive concentrations in Psychotria viridis, Psychotria carthagenensis, Banisteriopsis caapi, and Alicia anisopetala. Validation parameters were established; linearity (R2 = 0.988–0.999), limit of detection (LOD) (0.06–0.11 ng/mL), limit of quantitation (LOQ) (0.18–0.34 ng/mL), accuracy, precision, extraction efficiency (>98%), recovery (74.1–111.6%), and matrix effect (70.6–109%) were all evaluated. All six compounds eluted within nine minutes, with a total analysis time of 20 min including column equilibration. This method establishes a high-throughput method for the robust analysis of psychedelics which may see future use in agricultural research and industry.