The disease progression of rheumatoid arthritis (RA) is closely related to the disorder of amino acids metabolism. This study aimed to clarify the changes in the categories, quantities and metabolic pathways of amino acids associated with disease progression in adjuvant-induced arthritis (AIA) rats, and to evaluate the application value of amino acids metabolic profiling in the diagnosis of RA. A total of 20 rats were randomized into a control group and AIA model group. Thirty-three days after modeling, the synovial tissues of left ankle joints were collected for histopathological examination. Also, untargeted metabolomics based on UPLC-LTQ/Orbitrap MS was used to identify the potential amino acids and analyze the changes of their metabolic pathways. Histopathological observation showed that the synovial tissues of AIA rats exhibited hyperplastic and inflammatory lesions. Through multivariate statistical analysis, a total of 12 amino acids were identified and considered to show metabolic changes in AIA rats. Compared with the control group, the concentrations of glutamate, arginine, methionine were increased in the AIA model group; while the concentrations of proline, valine, tyrosine, phenylalanine, leucine, glycine, tryptophan, histidine, threonine were decreased. Metabolic pathway analysis showed that the selected amino acids had important physiological functions in immune and inflammatory response. The results suggested that amino acids could be employed as biomarkers closely related to RA, and the analysis of amino acids metabolic profiling also exhibited potential application value in the diagnosis, disease progression monitoring and therapy of RA.