Polyethylene glycol (PEG) represents an effective strategy to improve the pharmacokinetic profile of a molecule as it extends the biotherapeutic's half-life, masks immunogenic epitopes or modifies its distribution . The addition of one or multiple PEG moieties, in either linear or branched form, is known to carry the risk of potentially inducing an immunogenic response against PEG . The importance of accurately quantifying anti-PEG antibodies during a clinical study is well recognized and stems from the fact that anti-PEG antibodies have been shown to negatively impact the efficacy of the biotherapeutic that the PEG is coupled to . As a consequence, sponsors are encouraged to develop immunogenicity assays to assess appropriately the presence of anti-drug antibodies (ADA) against the protein component as well as the PEG . However, detection of anti-PEG antibodies is complicated by a number of technical challenges, including the availability of appropriate positive control material . In addition, the fact that some anti-PEG antibodies are known to circulate as low-affinity IgM, drives the need for an assay able to detect low affinity anti-PEG ADA even in the presence of high concentrations of the biotherapeutic . To address this need, we developed and validated an Affinity Capture Elution (ACE) -AGL assay to detect anti-drug and anti-PEG antibodies . In this assay, which we call ACE-AGL, ADA are captured by biotin-PEG-drug, acid eluted and re-captured on a second plate coated with protein AGL . ADA are then detected using Ruthenium-PEG-drug . The new assay format described is highly sensitive to both anti-drug and anti-PEG antibodies and very drug-tolerant . The ACE-AGL assay is easy to perform and has been successfully validated at two separate CROs . We propose the ACE-AGL format as a valid and effective alternative to the currently available assay methods.