Characterization of biopharmaceutical products is a challenging task, which needs to be carried out at several different levels (including both primary structure and conformation). An additional difficulty frequently arises due to the structural heterogeneity inherent to many protein-based therapeutics (e.g., extensive glycosylation or “designer” modifications such as chemical conjugation) or introduced postproduction as a result of stress (e.g., oxidation and deamidation). A combination of ion-exchange chromatography (IXC) with online detection by native electrospray ionization mass spectrometry (ESI MS) allows characterization of complex and heterogeneous therapeutic proteins and protein conjugates to be accomplished at a variety of levels without compromising their conformational integrity. The IXC/ESI MS measurements allow protein conjugates to be profiled by analyzing conjugation stoichiometry and the presence of multiple positional isomers, as well as to establish the effect of chemical modifications on the conformational integrity of each species. While mass profiling alone is not sufficient for identification of nonenzymatic post-translational modifications (PTMs) that result in a very small mass change of the eluting species (e.g., deamidation), this task can be completed using online top-down structural analysis, as demonstrated using stressed interferon-β as an example. The wealth of information that can be provided by IXC/native ESI MS and tandem mass spectrometry (MS/MS) on protein-based therapeutics will undoubtedly make it a very valuable addition to the experimental toolbox of biopharmaceutical analysis.