Oxygen radicals (such as hydroxyl radical) damage biological molecules and are likely to contribute to several pathophysiological conditions. Specific biomarkers of oxidative damage are needed to adequately assess the importance of oxidation in ageing and disease. Protein oxidation is now commonly measured by spectrophotometric detection of protein carbonyls; however, this technique is non-specific and does not necessarily indicate protein oxidation. Here we present a quantitative, sensitive method for the detection of oxidatively modified phenylalanine (i.e. 2-hydroxyphenylalanine and 3-hydroxyphenylalanine) in protein. The assay involves enzymatic hydrolysis of protein to release the constituent amino acids for derivatisation with pentafluorobenzyl bromide and subsequent extraction into n-decane. The di-O-pentafluorobenzyl derivatives of 2- and 3-hydroxyphenylalanine are resolved from other sample components using gas chromatography and detected with negative ion chemical ionization mass spectrometry. This procedure produces linear standard curves and sensitive detection limits for 2- and 3-hydroxyphenylalanine (25 and 5 fg on column, respectively). Stable isotope labelled 2- and 3-hydroxyphenylalanine (ring, d4) was synthesised and used in the assay as an internal standard to improve analytical precision. The assay was applied to determine the levels of 2- and 3-hydroxyphenylalanine in freshly isolated human serum and serum proteins. Exposure of protein to reactive oxygen species significantly increased the levels of 2- and 3-hydroxyphenylalanine as detected by the assay.