Peptides play a seminal role in most physiological processes acting as neurotransmitters, hormones, antibiotics, and immune regulation. In the context of tumor biology, it is hypothesized that endogenous peptides, hormones, cytokines, growth factors, and aberrant degradation of select protein networks (e.g., enzymatic activities, protein shedding, and extracellular matrix remodeling) are fundamental in mediating cancer progression. Analysis of peptides in biological fluids by mass spectrometry holds promise of providing sensitive and specific diagnostic and prognostic information for cancer and other diseases. The identification of circulating peptides in the context of disease constitutes a hitherto source of new clinical biomarkers. The field of peptidomics can be defined as the identification and comprehensive analysis of physiological and pathological peptides. Like proteomics, peptidomics has been advanced by the development of new separation strategies, analytical detection methods such as mass spectrometry, and bioinformatic technologies. Unlike proteomics, peptidomics is targeted toward identifying endogenous protein and peptide fragments, defining proteolytic enzyme substrate specificity, as well as protease cleavage recognition (degradome). Peptidomics employs "top-down proteomics" strategies where mass spectrometry is applied at the proteoform level to analyze intact proteins and large endogenous peptide fragments. With recent advances in prefractionation workflows for separating peptides, mass spectrometry instrumentation, and informatics, peptidomics is an important field that promises to impact on translational medicine. This review covers the current advances in peptidomics, including top-down and imaging mass spectrometry, comprehensive quantitative peptidome analyses (developments in reproducibility and coverage), peptide prefractionation and enrichment workflows, peptidomic data analyses, and informatic tools. The application of peptidomics in cancer biomarker discovery will be discussed.