Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl-4-hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 ± 3, and <2%, respectively; “top-down” MS/MS of these ions show oxidation at the sites R28–V31, E95–F107, and K216 of 22%, 28%, and 34%, respectively, but with a possible (∼4%) fourth site at V125–D150. However, for the doubly oxidized molecular ions (increasing the precursor oxygen content from 0.94 to 2), MS/MS showed an easily observable ∼13% oxygen at the V125–D150 site. For the “bottom-up” approach, detection of the ∼4% oxidation at the V125–D150 site by MS analysis of a proteolysis mixture would have been very difficult. The unmodified peptide containing this site would represent a few percent of the proteolysis mixture; the oxidized peptide not only would be just ∼4% of this, but the uniqueness of its mass value (∼1–2 kD) would be far less than the 11,933 Dalton value used here. Using different molecular ion precursors for top-down MS/MS also provides kinetic data from a single sample, that is, from molecular ions with 0.94 and 2 oxygens. Little oxidation occurs at V125–D150 until K216 is oxidized, suggesting that these are competitively catalyzed by the iron center; among several prolyl-4-hydroxylases the K216, H137, and D139 are conserved residues.