Oxidative stress is often the cause of a wide range of human chronic pathologies including but not limited to stroke, cardiovascular and neurodegenerative diseases. In this setting, increasing efforts are currently being made to the design and synthesis of new antioxidant compounds with enhanced efficacy. Among all the potential molecules of interest, synthetic nitrone spin-traps have attracted a great deal of research attention, particularly due to their dual function: effective inhibitors of oxidative stress and damage, and actual analytical tool for the detection and characterization of free radicals using Electron Paramagnetic Resonance (EPR) spectroscopy. In this study, two derivatives of benzoxazinic nitrones (3-aryl-2H-benzo[1,4]oxazin-N-oxides) bearing an electron-withdrawing methyl-acetate group on the benzo moiety (in para and meta positions respect to the nitronyl function) were synthesized. Their in vitro antioxidant activity was evaluated by means of the α,α-diphenyl-β-picrylhydrazyl radical (DPPH•) scavenging assay, and their inhibitory effects on the erythrocyte hemolysis induced by the water soluble free radical initiator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) compared. In addition, EPR was employed to monitor the decay profiles of DPPH• to evaluate the kinetic behavior of the different antioxidants tested. Results showed that the newly synthesized benzoxazinic derivatives, when compared to the unsubstituted parent compound, act as more effective antioxidants both in cell and cell-free systems. In particular, the para-substituted derivative was able to quickly scavenge DPPH to a greater extent in comparison with the meta-substituted isomer. Overall, these results clearly demonstrate that the introduction of an electron-withdrawing group in the phenyl moiety significantly increased the antioxidant capacity of benzoxazinic nitrones, thus showing exciting opportunities as new therapeutic agents in the treatment of diseases associated with oxidative stress.