Biotherapeutics must endure in-depth testing to validate their efficacy and safety before their release to the medical community. Characterization and quantitation of these large molecule medicines is traditionally performed with ligand binding assays or radiolabeling procedures. Issues with selectivity, accuracy, and unavailability of applicable assays for the characterization and quantitation of certain biotherapeutics means that liquid chromatography–mass spectrometry (LC–MS) is becoming an increasingly selected method for biotherapeutics testing. Typically used for small molecules, LC–MS can be adapted for larger molecule analysis with additional high throughput and multiplexing capabilities. New method development has turned LC–MS into a highly sensitive option for biotherapeutics validation.