Large-molecule therapeutics (e.g., peptides, proteins and oligonucleotides) are becoming increasingly prominent in the development pipelines of pharmaceutical companies. Bioanalysis of these molecules has historically required immunoassay approaches. However, recent advances in LC–MS instrumentation, combined with techniques developed in key applications such as proteomics, are now allowing LC–MS-based approaches for large-molecule bioanalysis. Industry interest is growing rapidly, as evidenced by the number of recent publications, workshops and symposia on the topic. In this chapter, we present a brief overview of current LC–MS-based approaches and considerations for bioanalysis of large-molecule therapeutics, primarily peptides and proteins, in the context of regulated drug development.