Nitrones have exhibited pharmacological activity against radical-mediated pathophysiological conditions and as analytical reagents for the identification of transient radical species by electron paramagnetic resonance (EPR) spectroscopy. In this work, competitive spin trapping, stopped-flow kinetics, and density functional theory (DFT) were employed to assess and predict the reactivity of O(2)(*-) and HO(2)(*) with various para-substituted alpha-phenyl-N-tert-butylnitrone (PBN) spin traps. Rate constants of O(2)(*-) trapping by nitrones were determined using competitive UV-vis stopped-flow method with phenol red (PR) as probe, while HO(2)(*) trapping rate constants were calculated using competition kinetics with 5,5-dimethylpyrroline N-oxide (DMPO) by employing EPR spectroscopy. The effects of the para substitution on the charge density of the nitronyl-carbon and on the free energies of nitrone reactivity with O(2)(*-) and HO(2)(*) were computationally rationalized at the PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level of theory. Theoretical and experimental data show that the rate of O(2)(*-) addition to PBN derivatives is not affected by the polar effect of the substituents. However, the reactivity of HO(2)(*) follows the Hammett equation and is increased as the substituent becomes more electron withdrawing. This supports the conclusion that the nature of HO(2)(*) addition to PBN derivatives is electrophilic, while the addition of O(2)(*-) to PBN-type compounds is only weakly electrophilic.