Reaction of the free-radical diphenylpicrylhydrazyl (DPPH˙) with Trolox® (TrOH) was investigated in buffered hydroalcoholic media by using a stopped-flow system. DPPH˙ was reduced to the hydrazine analogue DPPH-H with a measured stoichiometry of about 2. DPPH-H was characterized by an acid–base equilibrium (pK_a = 8.6). Time-resolved absorption spectra recorded with an excess of either TrOH or DPPH˙ indicated that no significant amount of the TrO˙ radical was accumulated. The TrO˙ radical formed in a first step further reacted quickly with DPPH˙. For 1 : 1 ethanol–buffer mixtures at pH 7.4, the bimolecular rate constants of the first and second steps were 1.1 × 10⁴ M⁻¹ s⁻¹ and 2 × 10⁶ M⁻¹ s⁻¹, respectively. A significant increase of the measured rate constant was observed for ethanol–buffer solutions as compared to ethanol. The rate was also increased at higher pH. A deuterium isotopic effect of 2.9 was measured. These data are discussed with regards to mechanisms involving either electron or proton exchange as rate determining steps in the reaction of DPPH˙ with Trolox®. The importance of solvent acidity control in investigation of antioxidant properties is outlined.