The oxidation of methionine is an important post-translational modification of proteins with numerous roles in physiology and pathology. However, the quantitative analysis of methionine oxidation on a proteome-wide scale has been hampered by technical limitations. Methionine is readily oxidized in vitro during sample preparation and analysis. In addition, there is a lack of enrichment protocols for peptides that contain an oxidized methionine residue, making the accurate quantification of methionine oxidation difficult to achieve on a global scale. Herein, we report a methodology to circumvent these issues by isotopically labeling unoxidized methionines with 18 O-labeled hydrogen peroxide and quantifying the relative ratios of 18 O-and 16 O-oxidized methionines. We validate our methodology using artificially oxidized proteomes made to mimic varying degrees of methionine oxidation. Using this method, we identify and quantify a number of novel sites of in vivo methionine oxidation in an unstressed human cell line.