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In response to DNA-damaging physical or chemical agents, the DNA damage repair (DDR) pathway is activated in eukaryotic cells. In the radiobiology field, it is important to assess the DNA damage effect of a certain irradiation regime on cancer cells and compare it to the effect on non-transformed cells exposed to identical conditions. The first step in the DNA repair mechanism consists of the attachment of proteins such as the phosphorylated histone γ-H2AX (p-γ-H2AX) to DNA double-strand breaks (DSB) in the nucleus, which leads to the formation of repairing foci. Therefore, imaging methods were established to evaluate the presence of foci inside the nucleus after exposure to DNA-damaging agents. This approach is superior in sensitivity to other methods, such as the comet assay or the pulsed-field gel electrophoresis (PFGE), that allow direct detection of cleaved DNA fragments. These electrophoresis-based methods require high ionizing radiation dosages and are difficult to reproduce compared to imaging-based assays. Conventionally, the number of foci is determined visually, with limited accuracy and throughput. Here, by exploring the effect of laser-plasma accelerated electrons FLASH irradiation on cancer cells, we describe an image cytometry protocol for the quantification of foci with increased throughput, upon large areas, with increased precision and sample-to-sample consistency. It consists of the automatic scanning of fluorescently labeled cells and using a gating strategy similar to flow cytometry to discriminate cells in co-culture based on nuclei elongation properties, followed by automatic quantification of foci number and statistical analysis. The protocol can be used to monitor the kinetics of DNA repair by quantification of p-γ-H2AX at different time points post-exposure or by quantification of other DNA repair proteins that form foci at the DNA DSB sites. Also, the protocol can be used for quantifying the response to chemical agents targeting DNA. This protocol can be performed on any type of cancer cells, and our gating strategy to discriminate cells in co-culture can also be used in other research applications. Key features • Analysis of DNA-damage sensitivity using model cancer cell lines and non-transformed cellular controls.• Allows comparative testing of various doses of DNA damaging radiation on cancer and non-transformed cells in co-culture, as well as in monocultures.• This protocol requires TissueFAXSiPlus model i12 or an alternative instrument that allows automatic image acquisition and stitching to benefit from enhanced analysis throughput.• For analyses of co-cultures or heterogeneous samples, TissueQuest software is required to selectively quantify different cell subpopulations; dedicated training is advisable before operating the system.