Biopharmaceuticals are drugs of biotechnological origin used as vaccines or for the treatment of non-communicable diseases, such as cancer or anemia. Due to their high efficacy and specificity, the market for novel and biosimilar biopharmaceuticals is growing immensely. This growth is accompanied by new challenges in quality control and analytical characterization during drug development and production. Glycosylation is one of the structural modifications that occur during production of many protein-based drugs and can have significant effects on their pharmacological properties. Mass spectrometry (MS) is a promising technique for high-quality analytical characterization of glycosylation, starting from early drug development through to final lot release. Here, we review the most recent trends in biopharmaceutical glycosylation analysis by MS with and without coupling to liquid chromatography or capillary electrophoresis, and draw comparisons with established, non-MS methods. We discuss future prospects for the emerging MS approaches for the biotech industry and biopharmaceutical research.