The inverse relationship between adalimumab concentration and ATA positive in serum samples from non-responding patients treated with adalimumab. Display omitted ► The authors describe the use of the homogeneous mobility shift assay (HMSA). ► HMSA was used to measure adalimumab and antibodies-to-adalimumab (ATA). ► The HMSAs for adalimumab and ATA have high sensitivity, precision and accuracy. ► Non-responding patients showed high incidence of ATA generation (44%) using HMSA. ► In non-responding patients, levels of adalimumab and ATA were inversely associated. This report describes the analytical validation and application of the homogeneous mobility shift assay (HMSA) method for the measurement of adalimumab and human antibodies-to-adalimumab (ATA) in serum samples from patients who have lost response to adalimumab treatment. Validation of the ATA- and the adalimumab-HMSA revealed a lower limit of detection to be 0.026U/mL for ATA and 0.018μg/mL for adalimumab in serum samples. Intra-assay and inter-assay precision determination yielded a coefficient of variation of less than 15%, and the accuracy of both assays was within 20%. Adalimumab drug tolerance in the ATA-HMSA was up to 20μg/mL in the test serum. Serum samples from 100 drug-naïve healthy subjects were tested to set-up the cut point of 0.55U/mL for ATA and 0.68μg/mL for adalimumab. Analysis of 100 serum samples from patients who were losing response to adalimumab showed that 26% had an adalimumab level below the cut point, of these 68% were ATA positive. Overall, 44% of the patients (44/100) were positive for ATA. This study presents evidence that drug and anti-drug antibody levels are important determinants of patient response to therapy.