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Antibiotic residues have the risk of promoting the emergence of resistant bacteria in the aquatic system. Gamma irradiation has been proven to be an effective technique to remove the contamination. However, degradation products might also promote antimicrobial resistance, especially when the active part of the parent structure remains unaltered. Therefore, degradation products have been analyzed in detail to evaluate their potential environmental effect. Levofloxacin lactate was employed as the targeted antibiotic to evaluate gamma radiolysis of antibiotics; the activity and cytotoxicity of the products were also analyzed. Levofloxacin lactate (10 μg mL−1) was removed >99% with the dose of 1 kGy. The G-values ranged from 1.643 to 0.193 with increasing radiation dose. Five main radiolysis products (mass/charge ratio [m/z] 346, 330, 318, 302, and 274) were identified by liquid chromatography-mass spectrometry (LC-MS). Results indicated degradation at the piperazinyl substituent and the quinolones m...