Immunopeptidomics is a mass spectrometry-based technique, crucial for characterizing the peptide repertoire displayed by Class I Human Leukocyte Antigens (HLA-I). This repertoire, known as the immunopeptidome, is central to adaptive immunity, as it facilitates T cell recognition of infected or malignant cells that present abnormal peptides. Understanding the immunopeptidome and the rules behind its generation is therefore essential for deciphering antigen presentation mechanisms and uncovering new diagnostic markers and therapeutic targets in infectious diseases, cancer and autoimmunity. This chapter outlines a simple immunopeptidomics protocol, adapted from established workflows with the vision to be accessible and to rely on equipment and reagents commonly available in most biochemistry laboratories, as well as open-source software. Crucially, it includes instructions for the in-house production and purification of the monoclonal antibody needed for HLA-I immunopeptidomics. Practical considerations and tips are also incorporated to empower less experienced researchers, allowing them to successfully apply immunopeptidomics to their own research questions.