Introduction: To prospectively evaluate quantitative assessment of fluorescence optical imaging (FOI) for differentiation of synovitic from non-synovitic joints in patients suffering from rheumatoid arthritis (RA). Methods: FOI of the hands was performed in patients with active RA, and a stratified quantitative fluorescence readout (FLRO) of 3 phases (1-120 s; 121-240 s; 241-360 s) was generated for 5 individual joints of the clinical predominant hand (carpal joint, metacarpophalangeal and proximal interphalangeal joints of digits II & III). To dissect the effect of the overall perfusion of the hand from the perfusion due to synovitis, a fluorescence ratio (FLRA) was additionally calculated, dividing each FLRO by the readout of the eponychium of digit II. The mean FLRO and FLRA were compared between joints with absent vs. present synovitis determined by clinical examination, grayscale, color Doppler ultrasonography, or magnetic resonance imaging (MRI). Results: The analysis for 90 individual joints from 18 patients yielded FLRO ranging from 4.4 to 49.0 x 10(3), and FLRAs ranging from 0.37 to 2.27. Overall, the analyses based on the FLRA revealed a higher discrimination than the analyses related to the FLRO, demonstrating most significant differences in phases 2 and 3. A sensitivity of 26/39 (67%) and a specificity of 31/40 (77%) were calculated for FLRA of phase 3 using a cut-off value of more than 1.2 to detect MRI-confirmed synovitis with FOI. Conclusions: FOI has a potential for visualizing synovitis in subjects with RA. For adequate FOI interpretation, quantitative analysis should be based on the novel FLRA calculated for phases 2 and 3.