E6011, a humanized anti-fractalkine monoclonal antibody, is under development for the treatment of various inflammatory diseases, such as rheumatoid arthritis. Therapeutic antibodies may induce production of anti-drug antibodies (ADA) that may deteriorate efficacy and/or enhance immunogenic reaction. It is important to have an ADA assay to understand the characteristics of biotherapeutics under development. A simple and reproducible assay has thus been developed for the determination of ADA against E6011 in monkey and human serum by electrochemiluminescence (ECL) detection. An immune-complex of biotinylated E6011, ADA, and ruthenium-labeled E6011 was attached to avidin-coated wells for ECL signal detection. Screening and confirmatory cutpoints were determined to judge negative or positive ADA. Sensitivity of ADA was 1.61 and 1.34 ng/mL in monkey and human serum, respectively. Accuracy and precision of the assay were within ±20% and 20%, respectively. Drug tolerance of the assay in monkey and human sera was ensured up to 100 and 1000 μg/mL E6011 at the surrogate ADA levels of 1 and 4 μg/mL, respectively. The developed assay was successfully applied to ADA quantification in monkeys and humans in support of immunogenicity assessments.