Biotherapeutics can provoke unwanted immunogenicity, which can affect the safety and/or efficacy of the drug. Consequently, immunogenicity risk assessments are carried out during drug development. Further, immunogenicity is evaluated during clinical trials prior to licensure. Most therapeutic proteins are either purified from plasma or, increasingly, manufactured using recombinant DNA technology. The immune response to these protein therapies involves internalization of the protein into antigen-presenting cells (APCs), enzymatic processing of the protein into peptides, and presentation of peptides on major histocompatibility class II (MHC II) proteins (HLA class II in humans). The MHC II–peptide complex if recognized by T cell receptors (TCRs) engages with CD4+ T helper cells, which ultimately results in formation of anti-drug antibodies (ADAs). The MHC-associated peptide proteomics (MAPPs) is an assay that in principle can identify the peptides derived from the therapeutic protein that are associated with an individual subject’s MHC variants. The MAPPs assay is the only assay that provides information about both protein processing in APCs and peptide presentation by MHC proteins. This is important because not all possible peptides from a protein can be generated during protein processing in APCs. Here we describe a highly robust protocol for MHC II MAPPs.