Tumour-associated cell surface markers are potential targets for antibody-based therapies. We have obtained a panel of myeloid cell binding single chain variable fragments (scFv) by applying phage display selection on myeloid cell lines followed by a selection round on freshly isolated acute myeloid leukaemia (AML) blasts using flow cytometry. To identify the target antigens, the scFv were recloned and expressed in an IgG(1) format and tested for their ability to immunoprecipitate cell surface proteins. The IgGs that reacted with distinct cell membrane extractable proteins were used in large-scale affinity purification of the target antigen followed by mass-spectrometry-based identification. Well-characterised cell surface antigens, such as leukocyte antigen-related receptor protein tyrosine phosphatase (LAR PTP) and activated leukocyte adhesion molecule (ALCAM) in addition to several unknown proteins, like ATAD3A, were identified. These experiments demonstrate that phage antibody selection in combination with affinity chromatography and mass spectrometry can be exploited successfully to identify novel antibody target molecules on malignant cells.