The characterization and absolute quantification of protein biopharmaceuticals and their product-related impurities, e.g., oxidation variants, is essential due to their potential impact on biological activity and immunogenicity. Here, we present site assignment and absolute quantification of oxidation variants of pegfilgrastim, a poly(ethylene glycol) modified recombinant human granulocyte-colony stimulating factor. Pegfilgrastim stressed with 1.0% hydrogen peroxide served as a model protein for developing a top-down high-performance liquid chromatography-mass spectrometry (HPLC-MS) platform that allowed direct site assignment of Met122, Met127, and Met138 oxidation within a total analysis time of 30 min. Three different absolute quantification methods, namely, UV absorption spectroscopy, full-scan MS, and all-ion fragmentation (AIF) MS were compared. Additionally, the monitoring of all generated fragment ions or selected sets of fragment ions were evaluated for the AIF method. Linearity of calibration curves from 5.0 to 25 ng μL(-1), 25 to 250 ng μL(-1), and 100 to 1000 ng μL(-1) was confirmed. The AIF method achieved a lower limit of detection of 0.85 ng μL(-1) and a lower limit of quantification of 2.54 ng μL(-1). On the basis of the comparison of relative standard deviations of interday measurements, AIF was concluded to be the method of choice for concentrations up to 50 ng μL(-1), and UV measurements should be carried out above this concentration. Finally, an expired pegfilgrastim batch was analyzed as a a real biopharmaceutical sample to confirm the feasibility of our approach for monitoring low levels of oxidation variants.