A simple, sensitive and precise spectrophotometric method has been developed for the determination of L-5-Hydroxytryptophan in Griffonia simplicifolia seed extracts and dosage forms. L-5-Hydroxytryptophan gives a blue colored chromogen with Folin-Ciocalteu reagent under alkaline conditions with absorption maxima at 736 nm. The chromogen obeys Beer’s law in the concentration range 1-10 g/ml. The proposed method is reproducible and statistically validated and recoveries range between 98.95% and 99.96%.