The electron spin resonance (EPR) spin-trapping technique allows detection of radical species with nanosecond half-lives. This technique is based on the high rates of addition of radicals to nitrones or nitroso compounds (spin traps; STs). The paramagnetic nitroxides (spin-adducts) formed as a result of reactions between STs and radical species are relatively stable compounds whose EPR spectra represent "structural fingerprints" of the parent radical species. Herein we report a novel protocol for the synthesis of N-tert-butylmethanimine N-oxide (EBN), which is the simplest nitrone containing an α-H and a tertiary α′-C atom. We present EPR spin-trapping proof that: (i) EBN is an efficient probe for the analysis of glutathione thiyl radical (GS·); (ii) β-cyclodextrins increase the kinetic stability of the spin-adduct EBN/·SG; and (iii) in aqueous solutions, EBN does not react with superoxide anion radical (O2-·) to form EBN/·OOH to any significant extent. The data presented complement previous studies within the context of synthetic accessibility to EBN and efficient spin-trapping analysis of GS.