The antigenic landscape of tumors is distinct from healthy cells and has been the rationale behind a variety of vaccination trials. Typically the target tumor-associated antigens have been of self origin and have rarely induced effective anti-tumor responses. Recent data show that activation of the immune system by immune checkpoint blocking therapies leads to tumor rejection and that recognition of mutated antigens, known as 'neo-antigens' plays a key role. Discovery of neo-antigens relies mainly on prediction-based interrogation of the 'mutanome' using genomic information as input, followed by T-cell screening. Recent breakthroughs in mass spectrometry (MS) based immunopeptidomics have allowed the discovery of very large pools of naturally presented peptides, among them neo-epitopes. This review highlights the current progress related to neo-antigens discovery with emphasis on prediction algorithms and MS as well as the synergy of the two methodologies and how they can be exploited to develop effective personalized immunotherapy.