In the present work we have developed a standard-addition HPLC method using a mobile phase containing low concentration of ZnAc(2) to determine physiological level of kynurenine (KYN), kynurenic acid (KYNA) and tryptophan (TRP) in human plasma simultaneously. The method greatly improved the sensitivity of KYNA, the resolution of KYNA and TRP, and avoided clotting risk caused by high concentration of ZnAc(2) in mobile phase. Samples were deproteinized by addition of equal volume of 0.6 mol/L HClO(4). Analytes in supernatants were separated by an Agilent HC-C18 (2) analytical column; an aqueous mobile phase containing 20 mmol/L NaAc, 3 mmol/L ZnAc(2) and 7% acetonitrile at flow rate of 1.0 mL/min. Detections were performed by a variable wavelength detector at wavelength 365 nm for KYN and a fluorescence detector at wavelengths excitation 344 nm and emission 398 nm for KYNA and TRP. Good linear responses were found with r(2)>0.999 for all analytes within the concentration range of physiological levels. The limit of detection of the developed method was 0.03 micromol/L, 0.9 nmol/L and 0.4 micromol/L for KYN, KYNA and TRP respectively. Recoveries from spiked human plasma were 95.4-99.7% for KYN, 98.9-104% for KYNA and 96.5-100.2% for TRP. All CVs for the repeatability and intermediate precision were less than 5%. We conclude that the developed method is helpful for the research investigations in KYN pathway of TRP metabolism.