There is growing interest in the therapeutic potential of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) for psychiatric disorders. Although 5-MeO-DMT can be chemically synthesized, the parotoid gland secretions of Incilius alvarius (also known as the Colorado River or Sonoran Desert toad) contain 5-MeO-DMT and other molecules including bufotenine, bufagenins, bufotoxins, and indole alkylamines that may have individual clinical utility or act as entourage molecules to enhance the activity of 5-MeO-DMT. I. alvarius is currently under severe ecological pressure due to demand for natural 5-MeO-DMT and habitat loss. We established a cell line from tissue obtained by wedge biopsy of the I. alvarius parotoid gland and confirmed the cell-based biosynthesis of 5-MeO-DMT by liquid chromatography with tandem mass spectrometry. Cellbased biosynthesis of I. alvarius parotoid gland secretions is a potentially cruelty-free and sustainable source of naturally derived 5-MeO-DMT for research and drug development.