With the technological advances of mass spectrometry (MS)-based platforms, clinical proteomics is one of the most rapidly growing areas in biomedical research. Urine proteomics has become a popular sub-discipline of clinical proteomics because it is an ideal source for the discovery of noninvasive disease biomarkers. The urine proteome offers a comprehensive view of the local and systemic physiology since the proteome is primarily composed of proteins/peptides from the kidneys and plasma. The emergence of MS-based proteomic platforms as prominent bioanalytical tools in clinical applications has enhanced the identification of protein-based urinary biomarkers. This review highlights the characteristics of urine that make it an attractive biofluid for biomarker discovery and the impact of MS-based technologies on the clinical assessment of urinary protein biomarkers.