Anti-drug antibodies (ADAs), specific for biotherapeutic drugs, are associated with reduced serum drug levels and compromised therapeutic response. The impact of ADA on the bioavailability and clin. efficacy of blockbuster anti-hTNF-α monoclonal antibodies is well recognized, especially for adalimumab and infliximab treatments, with the large and complex mol. architecture of classical Ig antibody drugs, in part, responsible for the immunogenicity seen in patients. The initial aim of this study was to develop solid-phase enzyme-linked immunosorbent assays (ELISA) and an in vitro cell-based method to accurately detect ADA and estimate its impact on the preclin. in vivo efficacy outcomes of two novel, nonimmunoglobulin VNAR fusion anti-hTNF-α biologics (Quad-X and D1-NDure-C4) and Humira, a brand of adalimumab. Serum drug levels and the presence of ADA were determined in a transgenic mouse model of polyarthritis (Tg197) when Quad-X and Humira were dosed at 1 mg/kg and D1-NDure-C4 was dosed at 30 mg/kg. The serum levels of the Quad-X and D1-NDure-C4 modalities were consistently high and comparable across all mice within the same treatment groups. In 1 mg/kg and 3 mg/kg Quad-X- and 30 mg/kg D1-NDure-C4-treated mice, an average trough drug serum concentration of 8μg/mL, 50μg/mL, and 350μg/mL, resp., were estimated In stark contrast, Humira trough serum concentrations in the 1 mg/kg treatment group ranged from <0.008μg/mL to 4μg/mL with trace levels detected in 7 of the 8 animals treated. Trough serum Humira and Quad-X concentrations in 3 mg/kg treatment samples were comparable; however, the functionality of the detected Humira serum was significantly compromised due to neutralising ADA. The impact of ADA went beyond the simple and rapid clearance of Humira, as 7/8 serum samples also showed no detectable capacity to neutralise hTNF-α-mediated cytotoxicity in a murine fibrosarcoma (L929) cell assay. The neutralisation capacity of all the VNAR constructs remained unchanged at the end of the exptl. period (10 wk). The data presented in this manuscript goes some way to explain the exciting outcomes of the previously published preclin. in vivo efficacy data, which showed complete control of disease at Quad-X concentrations of 0.5 mg/kg, equivalent to 10x the in vivo potency of Humira. This independent corroboration also validates the robustness and reliability of the assay techniques reported in this current manuscript, and while it comes with the caveat of a mouse study, it does appear to suggest that these particular VNAR constructs, at least, are of low inherent immunogenicity.