Cells are constantly exposed to a range of intrinsic and environmental factors, such as reactive oxygen species, ionizing radiation, and chemical agents, which threaten DNA integrity and can induce double-strand breaks (DSBs). DSBs are highly cytotoxic and must be rapidly repaired to maintain genome stability. One of the earliest chromatin marks associated with DSBs is the phosphorylation of histone variant H2A.X on serine 139, which leads to the formation of γ-H2A.X foci detectable by immunofluorescence. Immunocytochemical detection of γ-H2A.X is a widely used method to indirectly assess DSB formation and repair. In this chapter, we describe a simple and efficient immunofluorescence (IF) procedure for detecting DSB formation in U-2 OS cells treated with genotoxic agents. This technique does not require an expensive laboratory setup and can also be used to detect the formation of foci of additional repair factors using appropriate labeling.