Context: Mediumship is the alleged ability to communicate with deceased personalities. Previous studies have suggested that the endogenous psychotomimetic molecules bufotenine (BT) and dimethyltryptamine (DMT) may play a role in the pathogenesis of psychotic disorders. Distortion of perceptions observed during spiritual experiences could supposedly relate to these substances. Objective: To compare the presence of BT and DMT in human urine samples between individuals with and without mediumistic experiences. Methods: All participants (5 from medium's group - MG and 5 from non-medium's group - CG) undertook a single night continuous 6-h urine pool collection (6:00–11:59 PM). Mediums collected urine samples in nights when they reported having experienced mediumistic communication. A sensitive high-performance liquid chromatography-mass spectrometry (HPLC-MS) assay was used. Questionnaires were used to detect common mental disorders symptoms, and to screen and quantify anomalous experiences. Results: DMT was not detected in any urine specimen tested. The presence of BT detection in urine samples was greater in CG (2/5) than in MG (1/5), with no significant differences (p > 0.99). MG reported more anomalous experiences than CG (6.6±0.8 vs. 2.2±1.5, p = 0.03), but there was no difference concerning their mental health. Conclusion: There were no differences between individuals with and without alleged mediumistic experiences concerning endogenous psychedelics. Both BT and DMT are highly sensitive to metabolism by monoamine oxidase and to N-oxidation, and do not survive in the periphery for long. Alternative strategies should be considered to further investigate the putative role of the endogenous psychedelics pathway for the spiritual experiences.