All organisms contain 1,000s of proteins and peptides in their body fluids, which undergo disease-specific changes. Advances in the understanding of the functional relevance of these polypeptides under different (patho)physiological conditions and the identification of indicative changes with disease would greatly enhance diagnosis and therapy. The low-molecular-weight proteome, also termed peptidome, provides a rich source of information. Due to its lower molecular weight, the peptidome can be assessed without the need for sample manipulation like tryptic digests. This advantage facilitates comparative analysis but it also raises technical challenges differing from those in proteomics. The first part of this manuscript, is focused on the low-molecular-weight urinary proteome and reviews methodological aspects of sample collection, preparation, analysis, and data evaluation. The second part summarizes the recent progress in the definition and identification of clinically relevant polypeptide markers.