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The use of alpha particles irradiation in clinical practice has gained interest in the past years, for example with the advance of radionuclide therapy. The lack of affordable and easily accessible irradiation systems to study the cell biological impact of alpha particles hampers broad investigation. Here we present a novel alpha particle irradiation set-up for uniform irradiation of cell cultures. By combining a small alpha emitting source and a computer-directed movement stage, we established a new alpha particle irradiation method allowing more advanced biological assays, including large-field local alpha particle irradiation and cell survival assays. In addition, this protocol uses cell culture on glass cover-slips which allows more advanced microscopy, such as super-resolution imaging, for in-depth analysis of the DNA damage caused by alpha particles. This novel irradiation set-up provides the possibility to perform reproducible, uniform and directed alpha particle irradiation to investigate the impact of alpha radiation on the cellular level.