Double-strand breaks (DSBs) are highly deleterious DNA lesions because they can lead to chromosome aberrations or apoptosis. Various physical, chemical, and biological factors are involved in DSB induction. The formation of nuclear DSBs triggers phosphorylation of H2AX at Ser139; phosphorylated H2AX is named gamma H2AX. It is believed that histone H2AX phosphorylation is required for the concentration of DNA repair proteins to the damaged chromatin. H2AX is phosphorylated by members of phosphoinositide 3-kinase-related protein kinases (PIKKs), such as ATM (ataxia teleangiectasia mutated), which is the main mediator of H2AX phosphorylation in response to DSB induction. The development of immunocytochemical methods of gamma H2AX detection provided a convenient tool for research and is considered a gold standard for DSB analysis. These methods are sensitive and specific in the detection of a single DSB. Assessment of H2AX phosphorylation can be used in clinical practice as a marker of premalignant lesions and to predict cell sensitivity to radiotherapy and chemotherapy.