Abstract: Objective: To establish a LC- MS / MS method for the determination of tryptophan( TRP) 5- hydroxy-tryptophan( 5- HTP),5- hydroxytryptamine( 5- HT) and 5- hydroxyindole acetic acid( 5- HIAA) in mouse brain tissues.Method: Chromatographic separation was performed on a TSK Gel amide 80 column( 2.0 mm × 15 cm,3 μm) at column temperature of 35 ℃. The mobile phase system was a mixture of acetonitrile- ammonium formate solution( 15 mmol·L- 1,p H = 5.5)( 40 ∶ 60) whereas the flow rate was set at 0.4 m L·min- 1. The instrument was operated under the multiple reaction monitoring( MRM) mode using electrospray ionization( ESI) in the positive ion mode.Results: Four analyses were separated within 5 minutes. The lowest limits of quantification in tryptophan( TRP) 5-hydroxy-tryptophan( 5-HTP),5- hydroxytryptamine( 5- HT) and 5- hydroxyindole acetic acid( 5- HIAA)were 0.2,0.1,0.2 ng·m L- 1and 0.2 ng·m L- 1,respectively.The minimum detection limit were 0.05,0.02,0.05 ng·m L- 1and 0.05 ng·m L- 1,respectively.A good linear relationship was achieved within the concentration ranges from 1 to500 ng·m L- 1for the analyses.Precision and recovery of this method were in line with the requirements of biological sample analysis. The method was applied to determine the concentrations of four analyses in brain after oral administration of Hemerocallis flavonoid extract and results showed that four kinds of analyses were significantly changed which was similar with the positive drug of paroxetine. Conclusion: This method was simple,accurate,highly sensitive,strongly,specific and repeatable which indicated that was suitable for the determination of fourmetabolites of tryptophan-5- HT metabolic pathways in mice. This method could be used to the pharmacological mechanism of the neuropsychiatric drugs.