A high-performance liquid chromatography method with a diode array and an electrochemical detection (HPLC-ECD/DAD) was developed to determine the levels of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in rat plasma. The prepared samples were separated on a BDS column (4.6mm×250mm, 5mm) with column oven temperature of 25°C. The mobile phase consisted of 5% acetonitrile and a buffer solution, which contained 25mmol/L sodium acetate and 0.01mmol/L EDTA, adjusting pH to 4.5 with acetic acid, and it was pumped at a flow-rate of 1.0mL/min. KYN and KYA were measured by a variable wavelength detector at wavelengths 360nm and 333nm respectively, TRP and vanillic acid (as IS) both were measured at 280nm. Determination of 5-HT and 5-HIAA was accomplished at the electrochemical working potential of 700mV. Total run time was 14min. Several parameters of the developed method were validated including linearity, accuracy precision, and stability. The results showed the established method had good LOD and separation for all of the five compounds and IS in the biological matrix. The method is simple, fast, economical and accurate. The analytical method and the results could provide a reference for the clinical and scientific research of depression.