The identification of targetable antigens is crucial for advancing cancer immunotherapy. While traditional methods focus on tumor-associated antigens and somatic variants, recent advancements have highlighted novel antigen classes such as splice isoforms, gene fusions, ribonucleic acid (RNA) editing, and novel or unannotated open reading frames (nuORFs). These novel antigens offer promising targets due to their potential abundance and immunogenicity but are typically missed by traditional methods. Ribosome profiling (Ribo-seq) has emerged as a powerful technique for assessing and verifying translation of nuORFs that offer a plethora of novel targets. Mass spectrometry (MS)-based proteomics can validate the presence of translated proteins and peptides identified by Ribo-seq, providing direct evidence of protein expression and enhancing antigen discovery, potentially leading to the identification of novel cancer antigens for the development of more effective immunotherapies. This chapter explores the methodologies, challenges, and applications of Ribo-seq in combination with proteogenomic approaches for antigen discovery.