In recent years the breakthrough clinical success of T-cell based immunotherapy approaches including advances in allogeneic stem cell transplantation, checkpoint inhibitors, CAR T cells, bispecific antibodies and adoptive T-cell transfer have revolutionized treatment of several hematological malignancies (HM). However, still some patients do not respond to available therapeutic strategies at all, others for limited time only. Remaining challenges to augment efficacy of T cell-based immunotherapy are particularly to improve specificity and increase the frequency of anti-tumor immune responses, to reduce toxicity and to expand the spectrum of targetable cancer entities. A rational and promising approach to achieve this goal is peptide-based immunotherapy, which represents a low side-effect approach relying on specific immune recognition of tumor-associated HLA (human leucocyte antigen) presented peptides. In the recent years, our junior research group focused on the identification and characterization of such tumor-associated antigens for hematological malignancies using the approach of direct isolation and mass spectrometric analysis of HLA-presented peptides on the cell surface of leukemia cells. Using this approach we were able identify more than 45,000 HLA class I and II ligands representing more than 10,000 source proteins in acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). To identify broadly represented leukemia-associated antigens that can serve as targets for peptide-based immunotherapy, we developed a novel platform that defines tumor-associated antigens (TAA) based on their exclusive and high frequent representation in the immunopeptidome of HM patients. Hereby we are able to identify several novel, immunogenic leukemia-associated antigens, which are further validated to be broadly and frequently represented across different stages and mutational subtypes, remain stable under standard treatment and are targets for spontaneous memory T-cell responses in HM patient. Based on our data for CLL we have already establish the first multi-center, patient-individualized peptide vaccination trial for CLL patients, which is successfully recruiting since 10/2016 (iVAC-L-CLL01, NCT02802943).