"nBackground and the purpose of the study:Melatonin has recently been found in several plant tissues. Some reports show that the majority of the herbs containing the high level of melatonin have been used traditionally to treat neurological disorders or diseases associated with the generation of free radicals. Current study was undertaken to screen some medicinal plant species with historical evidence of efficacy in the treatment of neurological and antioxidant deficiency related disorders for their melatonin content. The melatonin content of boiled and alcoholic extracts were also compared. "nMethods: In this study, three medicinal herbs, Tanacetum parthenium (L.) Schultz. Bip. (Asteraceae), Tripleurospermum disciforme (C.A.Mey) Schultz. Bip. (Asteraceae) and Viola odorata (L.) (Violaceae) were analyzed using high performance liquid chromatography with ultraviolet detector (HPLC-UV), enzyme linked immunosorbent assay (ELISA) and thin layer chromatography (TLC). "nResults: Melatonin content in the dry plant powders differed with different assay methods (p < 0.001). For example, the melatonin content in T. disciforme was determined as 3.073 μg/g and 2.906 μg/g by the HPLC and the ELISA methods, respectively. Major conclusion:The results demonstrated that a hydroalcoholic solution could extract more melatonin from flowers of the herbs than hot water (p < 0.001). The presence of melatonin in these plant tissues may provide some explanation for the anecdotal evidence of their physiological effects in humans.