A reversed phase HPLC method for separation and determination of the major tryptophan (TRP) metabolites in both pyrrolas pathway and TRP hydroxylase pathway, including TRP, kynurenine(KN), 3-hydroxykynurenine(3-HKN), kynurenic acid(KA), xanthurenic acid(XA), 5-hydroxytryptophan(5-HTP), 5-hydroxytryptamin(5-HT) and 5-hydroxyindoleacetic acid(5-HIAA), has been developed by sequential optimization of mobile phase based on acetate buffer and methanol. Trichloroacetic acid(TCA) was used as ion-pairing reagent to increase the retention of 3-HKN. The effects of pH and concentrations of TCA on separation were studied. Good separation can be achieved at pH 4.0-5.0 of mobile phase in less than 25 min. When TCA was not used, 3-HKN was hard to be detected in biological samples. The maximum retention of 3-HKN was obtained at pH 4.0 with mobile phase containing 50 mmol/L TCA. Combination of electrochemical (ED) and ultraviolet (UV) detection was used in which ED was responsible for detection of 3-HKN, 5-HTP, 5-HT and 5-HIAA and UV for the others. The influences of potential of ED and wavelength of UV on detection were studied. The optimization of conditions for separation and detection in different biological samples was also discussed.