PEGylated proteins are a rapidly growing class of biopharmaceutical products, but their analytical characterization remains a formidable problem due to the extreme heterogeneity of these species. While significant advances have been made in recent years in this field due to integration of mass spectrometry in the analytical workflow, quick identification of PEGylation sites remains an unmet goal, particularly if several isoforms of the protein–polymer conjugate are present in the sample. To achieve this objective, a new method is developed, which utilizes a combination of ion exchange chromatography and top-down mass spectrometry consisting of two consecutive fragmentation steps (MS 3 ) to identify various conjugates. The method is tested with a complex mixture of products of ubiquitin conjugation with 5kDa PEG.