AIM: Monitoring immune responses to therapeutic peptides with endogenous counterparts is crucial for evaluating drug safety and efficacy. In this paper, we focused on the selection of an optimal assay format to develop a sensitive, robust, and drug-tolerant immunoassay for the detection of anti-drug antibody (ADA) against a therapeutic peptide. RESULTS: We assessed distinct ADA assay formats for preclinical and clinical studies, such as direct binding with labeled protein A/G, direct binding with labeled multiple species-specific antibodies for detection, bridging and affinity capture elution (ACE) formats. The assay formats were evaluated based on multiple assay parameters including sensitivity, drug tolerance, individual matrix variability and inter-assay precision. Overall, direct binding assay with labeled protein A/G for detection, which utilized less labeled peptide drug and achieved desired sensitivity and drug tolerance, is appropriate for preclinical studies. Bridging assay is more suitable format to support clinical studies as bridging assay has less assay variability than ACE assay. CONCLUSION: This study highlighted advantages and limitations of each ADA assay format for peptide drugs and evaluated the performance of different assay formats in the assay development process to aid in the selection of the best fit-for-purpose assay formats for preclinical and clinical phases.