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Gamma irradiation is a useful technology for degrading mycotoxins. The purpose of this study was to investigate the effect of irradiation on ochratoxin A (OTA) stability under different conditions. OTA was irradiated in methanolic solution and on millet flour at doses of 2 and 4 kGy. Residual OTA concentrations and possible degradation products in irradiated samples were analyzed by high-performance liquid chromatography with fluorescence detection and liquid chromatography coupled to mass spectrometry. The extent of in vitro cytotoxicity of OTA to HepG2 cells, with and without irradiation treatment, was assessed with an MTT assay. OTA was more sensitive to gamma radiation on Tunisian millet flour than in methanolic solutions. After irradiation of naturally contaminated millet flour, the OTA concentration was significantly reduced by 48 and 62% at a dose of 2 and 4 kGy, respectively. However, in the methanolic solution, OTA at concentrations of 1 and 5 μg mL−1 was relatively stable even at a dose of 4 kGy, with no degradation products detected in the chemical analysis. Analytical results were confirmed by cell culture assays. The remaining cytotoxicity (MTT assay) of OTA following irradiation was not significantly affected compared with the controls. These findings indicate that gamma irradiation could offer a solution for OTA decontamination in the postharvest processing chain of millet flour. However, the associated toxicological hazard of decontaminated food matrices needs more investigation.