There is growing interest in the development of new methods for elucidating the biological effects of low-dose exposure to ionizing radiation (IR) on human health. Herein, we introduce a strategy for assessment of the impact of radiation-induced oxidative stress on the intra-cellular metabolism of human leukocytes. Untargeted metabolomic analyses were performed by CE-ESI-MS on irradiated leukocytes exposed to increasing doses of -radiation emitted from a Taylor source, which were subsequently incubated for 44 h to allow for cellular recovery. Flow cytometry with dual fluorescence staining revealed a major shift from early- to late-stage apoptosis associated with cell membrane permeability changes as radiation dosage was increased relative to the control, but with a significant attenuation measured at intermediate dose levels. CE-ESI-MS analysis of filtered white blood cell lysates was also performed to quantify changes associated with 22 intra-cellular metabolites, which were consistently measured in leukocytes across all radiation levels. Preliminary experiments demonstrated that there was an overall depletion in metabolites with extended exposure to IR; however, there was a non-linear upregulation of specific metabolites at the 4 Gy level relative to pre-irradiated levels, notably for arginine, glutamine, creatine, proline and reduced glutathione. Our studies demonstrate that leukocytes require a minimum threshold level of IR to induce a cytoprotective response in metabolism associated with antioxidant defense, energy homeostasis and cell signaling, which is relevant to improved understanding of the mechanisms of oxidative stress in radiobiology and cancer therapy.