5-Hydroxytryptophan (1) is a naturally occurring amino acid found in significant levels in seeds of Griffonia simplicifolia and used in the treatment of the numerous effects of serotonin deficiency syndrome. An HPLC method has been developed for the direct assay of 1 in seeds of G. simplicifolia which overcomes the problems associated with previous techniques. By optimising the solvent extraction procedures and the HPLC conditions, levels of 1 could be estimated following a single-step seed extraction. The chromatographic conditions, solvent system and the extraction technique developed make this method relatively simple, fast and efficient. Using the described methods, the highest ever levels of 1 (namely, 20.83% on a fresh weight basis) have been determined in seeds of G. simplicifolia obtained in Ghana.