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Immunogenicity testing for antidrug antibodies (ADA) faces challenges when high levels of the drug are present in clinical patient samples. In addition, most functional cell-based assays designed to characterize the neutralizing ability of ADA are vulnerable to interference from endogenous serum components. Bead extraction and acid dissociation (BEAD) has been successfully applied to extract ADA from serum samples prior to conduction of cell-based assays. However, in the BEAD, certain amounts of the drug and endogenous serum components (so-called residual drug and serum components) from serum samples are carried over to final BEAD eluates due to formation of protein complexes with ADA or nonspecific binding with the beads. Using current enzyme-linked immunosorbent assay (ELISA)-based ligand-binding assays, it is difficult to evaluate the residual drug, which is complexed with excessive amounts of ADA and endogenous serum components in the BEAD eluates. Here, we describe an innovative application of LC-MS/MS for simultaneous detection of the residual human monoclonal antibody drug and endogenous human IgG and the neutralizing antibody positive-control (NAb-PC) in the BEAD eluates. In this study, the low levels of the residual drug and human IgG in the BEAD eluates indicate that the BEAD efficiently removed the high-concentration drug and serum components from the serum samples. Meanwhile, the NAb-PC recovery (∼42%) in the BEAD provided an acceptable detection limit for the cell-based assay. This novel application of LC-MS/MS to immunogenicity assay development demonstrates the advantages of LC-MS/MS in selectivity and multiplexing, which provides direct and fast measurements of multiple components for immunogenicity assay development.