Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease and derived from immune granule cells. It has been suggested playing an important role in the development of rheumatoid arthritis. In this study, a coumarin based fluorescent probe (GF-AFC) was designed and synthesized to evaluate DPPI activity in serum or tissue homogenates of collagen-induced arthritis (CIA) rats, an inflammatory arthropathy model. It was revealed that the fluorescent intensity was significantly increased in a very short time after specific substrate GF-AFC reacted with the DPPI. The fluorophore (AFC) was released to shine after the cleavage reaction which was examined by F-19 NMR spectroscopy. It has been shown that DPPI hydrolyzed the GF-AFC in a robust, linear, and time dependent manner at a significant high rate. A serum-based DPPI activity assay was validated by spiking and gradient dilution methods, there were no interferences or auto-fluorescence observed. The Coefficient of Variance calculated for serum-based DPPI activity assays indicates the good reproducibility. The good correlation has been seen between serum DPPI levels and the severity of arthritis during RA development in CIA rats. Our study has demonstrated a new serum based diagnostic assay for detecting DPPI activity using coumarin conjugated fluorescent (GF-AFC) as a substrate. The successful implementation of the case would provide beneficial experience in rheumatoid arthritis research.(C) 2016 Elsevier Ltd. All rights reserved.