Updated publication reference for PubMed record(s): 34798331. Updated publication reference for PubMed record(s): 34798331, 34723952. Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in a MS-based immunopeptidomic workflow, sample preparationis critically important for capturing high-quality data of therapeutic relevance. Here, we describe step-by-step instructions to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control (QC) samples. Our QC samples are MHC class I and class II peptides isolated from mouse (EL4 and A20) and human (JY) cell lines. We thoroughly describe the various reagents and specfic antibodies that are used too isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. QC samples generated using the described protocol can be used to establish and standardize immunopeptidomic workflow, as well as to benchmark new protocols. We anticipate that the visualized protocol provided in this report will represent a great starting point for any non-expert in addition to foster the instra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.