Exposure to a drug at the site of inflammation may be an important consideration for the effective treatment of inflammatory disorders such as rheumatoid arthritis (RA). The purpose of this in vivo study was to identify a methodology to enable effective quantification of antibody-type reagents in normal and inflamed tissue by investigating the distribution of the tumor necrosis factor-alpha (TNF-alpha) inhibitors, certolizumab pegol, adalimumab, and infliximab, in healthy and inflamed murine tissue using a novel non-invasive biofluorescence method. Certolizumab pegol, adalimumab, and infliximab were labeled with the low molecular weight dye alexa680. The agents were administered intravenously at a dose of 2mg/kg in naïve DBA/1 mice and in DBA/1 mice with ongoing collagen-induced arthritis. Concentrations of the TNF inhibitors in the hind paws were measured using a Xenogen IVIS200 biofluorescence imager at multiple time points up to 26h post-administration. In 2 independent experiments, the distribution of certolizumab pegol was compared with that of adalimumab and infliximab. Certolizumab pegol, adalimumab, and infliximab all distributed more effectively into inflamed tissue than non-inflamed tissue in this animal model of arthritis. However, the ratio of penetration of certolizumab pegol into inflamed arthritic paws compared with normal tissue was greater than that observed with adalimumab and infliximab. Furthermore, the duration of exposure in the inflamed versus normal tissue was more prolonged for certolizumab pegol than for both adalimumab and infliximab, and the accumulation of certolizumab pegol in diseased tissue was more responsive to the severity of inflammation when compared with adalimumab and infliximab. It is probable that these features of certolizumab pegol are conferred on the molecule by PEGylation. It is important to assess exposure to drug at the site of inflammation, because distinct structural features of certain agents may affect efficacy, tolerability, rapidity and/or sustainability of effect. The novel non-invasive biofluorescence method used in this study is an effective tool for comparing tissue penetration of therapeutic agents.