Ayahuasca is a psychoactive beverage prepared traditionally from a mixture of the leaves and stems of Psychotria viridis and Banisteriopsis caapi, respectively, being originally consumed by indigenous Amazonian tribes for ritual and medicinal purposes. Over the years, its use has spread to other populations as a means to personal growth and spiritual connection. Also, the recreational use of its isolated compounds has become prominent. The main compounds of this tea-like preparation are N,N-dimethyltryptamine (DMT), β-Carbolines, and harmala alkaloids, such as harmine, tetrahydroharmine, and harmaline. The latter are monoamine-oxidase inhibitors and are responsible for DMT psychoactive and hallucinogenic effects on the central nervous system. Although consumers defend its use, its metabolic effects and those on the central nervous system are not fully understood yet. The majority of studies regarding the effects of this beverage and of its individual compounds are based on in vivo experiments, clinical trials, and even surveys. This paper will not only address the toxicological aspects of the ayahuasca compounds but also perform a comprehensive and critical review on the analytical methods available for their determination in biological and non-biological specimens, with special focus on instrumental developments and sample preparation approaches.