Recent studies have suggested that visualization of γ-H2AX nuclear foci can be used to estimate exposure to very low doses of ionizing radiation. Although this approach is widely used for various purposes, its suitability for individual human biodosimetry has not yet been assessed. We therefore conducted such an assessment with the help of available software for observing and automatically scoring γ-H2AX foci. The presence of γ-H2AX foci was evaluated in human peripheral blood lymphocytes exposed ex vivo to γ rays in a dose range of 0.02 to 2 Gy. We analyzed the response of γ-H2AX to ionizing radiation in relation to dose, time after exposure, and individual variability. We constructed dose–effect calibration curves at 0.5, 8 and 16 h after exposure and evaluated the threshold of detection of the technique. The results show the promise of automatic γ-H2AX scoring for a reliable assessment of radiation doses in a dose range of 0.6 Gy to 2 Gy up to 16 h after exposure. This γ-H2AX-based assay may be useful for biodosimetry, especially for triage to distinguish promptly among individuals the ones who have received negligible doses from those with significantly exposures who are in need of immediate medical attention. However, additional in vivo experiments are needed for validation.