Background:To optimize treatment of inflammatory diseases, interest in the measurement of anti-tumor necrosis factor alpha (anti-TNF) serum drug concentrations is increasing. Preferably, assays for the detection of these drugs should be compared using the same reference material. In this study, 2 commercially available enzyme-linked immunosorbent assays (ELISAs) and a commercially available bioassay for the determination of anti-TNF drugs are compared.Methods:Serum samples from infliximab-, adalimumab-, and etanercept-treated patients, control samples from ustekinumab-treated patients, and healthy donors were obtained. ELISAs manufactured by Sanquin and Theradiag and the iLite reporter gene-based bioassay from Biomonitor were compared.Results:Sanquin, Theradiag, and iLite assays concordantly (100%) detected infliximab, adalimumab, and etanercept in the relevant patient groups. The Sanquin ELISAs specifically detected the anti-TNF drug they were designed for, whereas the Theradiag and iLite showed cross-reactivity with other anti-TNF drugs. Ustekinumab was not detected in any of the assays. Sanquin, Theradiag, and iLite exhibited linear quantitative correlation for all drug concentration assays. However, there were statistically significant quantitative differences in measured concentrations.Conclusions:All 3 commercially available assays seem suitable for therapeutic drug monitoring of anti-TNF drugs, allowing sensitive and comparable detection of infliximab, adalimumab, and etanercept concentrations, however with differences in specificity and recovery.