This updated edition explores essential techniques and newly emerged applications used in the expanding field of peptidomics. The opening chapters describe sample preparation and basic techniques. Subsequent chapters delve into quantitative peptidomics, bioinformatics, deep learning approaches, non-human model organisms, and peptide toxins. The last section includes multiple chapters on applications of peptidomics for human clinical specimens. Written for the highly successful Methods in Molecular Biology series, chapters include overviews and introductions to their respective topics, lists of the necessary materials, reagents, and instrumentation, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.