A single-dose cynomolgus monkey pharmacokinetic study was performed comparing two monoclonal anti-TNF antibodies (mAbs), GNExTNFvF and Humira. Normal pharmacokinetic profiles were observed over the first week of the study, followed by a rapid drop in serum mAb levels after day 8. In order to determine whether an anti-therapeutic antibody (ATA) response led to the abnormal clearance of antibody in this study, ATA assays were developed using two electrochemiluminescent technologies, BioVeris and Meso Scale Discovery (MSD). Characterization of the assays demonstrated that the two platforms gave similar sensitivities and tolerance to the presence of therapeutic antibody. Analysis of the cynomolgus monkey serum samples revealed that all animals developed significant ATA titers with log titer values of 2-4, with the BioVeris and MSD technologies giving very similar results. Immunodepletion studies confirmed the CDR-specificity of the ATA response for the GNExTNFvF-dosed cynos, although the Humira-dosed cynos showed both CDR-specific and human IgG1 framework-specific ATAs. To further characterize the ATA response, neutralizing antibody (NAb) assays were developed using two different approaches, flow cytometry and MSD. Flow cytometry and MSD cell-binding assays used Jurkat cells transfected with noncleavable TNF (huTNF(NC)). Neutralizing activity was assessed by the ability of ATA-positive serum samples to block the binding of biotinylated anti-TNF to huTNF(NC) Jurkat cells, showing that all but one animal developed neutralizing antibodies. Although both technologies displayed similar trends, the MSD approach showed greater differentiation between samples and could detect a broader range of neutralizing activities.