The primary objective of this review is to provide a comprehensive overview of the challenges involved in detecting ancient ayahuasca, a traditional hallucinogenic drink from the Amazon region, which is prepared using Banisteriopsis caapi liana and other plants, by utilizing advanced analytical techniques. The presence of harmine and harmaline in Andean archaeological findings has led certain authors to speculate that the Banisteriopsis liana may have been the source plant responsible for their occurrence. Consequently, the utilization of this liana can be traced back to at least 500 CE. However, a combination of archaeological, ethnobotanical, biochemical and analytical chemistry considerations has rendered uncertain the true origins of the presence of harmine and harmaline in ancient mummies and artifacts. Thus, the archaeological evidence does not convincingly prove the ancient usage of ayahuasca.