AIM: To determine the differences of amino acid (AA) levels in experimental autoimmune uveoretinitis (EAU). METHODS: AA analysis of the plasma samples in EAU rats induced by interphotoreceptor retinoid-binding protein emulsion were performed with high performance liquid chromatography (HPLC) and phenylisothiocyanate (PITC) pre-column derivation methods were performed. Using partial least squares discriminant analysis (PLS-DA), the potential biomarkers were identified in EAU rat plasma, and the metabolic pathways related to EAU were further analyzed. RESULTS: The method results showed that linear (r≥0.9957), intra-day reproducible [relative standard deviation (RSD)=0.04%-1.33%], inter-day reproducible (RSD=0.06%-2.07%), repeatability (RSD=0.03%-0.89%), stability (RSD=0.05%-2.48%) and recovery (RSD=1.98%-4.39%), with detection limits of 0.853-11.4 ng/mL. The metabolic profile in EAU rats was different from that in the control groups five AAs concentrations were increased and nine AAs were reduced. Moreover, five metabolic pathways were related to the development of EAU. CONCLUSION: The developed method is a simple, rapid and convenient for determination of AAs in EAU rat plasma, and these findings will provide a comprehensive insight on the metabolic profiling of the pathological changes in EAU.