Modern medicine has experienced a tremendous explosion in knowledge about disease pathophysiology, gained largely from understanding the molecular biology of human disease. Recent advances in mass spectrometry and proteomics now allow for simultaneous identification and quantification of thousands of unique proteins and peptides in complex biological tissues and fluids. In particular, proteomic studies of urine benefit from urine's less complex composition as compared to serum and tissues, and have been used successfully to discover novel markers of a variety of infectious, autoimmune, oncological, and surgical conditions. This perspective discusses the challenges of such studies that stem from the compositional variability and complexity of human urine, as well as instrumental sampling limitations and the effects of noise and selection bias. Strategies for the design of observational clinical trials, physical and chemical fractionation of urine specimens, mass spectrometry analysis, and functional data annotation are outlined. Rigorous translational investigations using urine proteomics are likely to discover novel and accurate markers of both rare and common diseases. This should aid the diagnosis, improve stratification of therapy, and identify novel therapeutic targets for a variety of childhood and adult diseases, all of which will be essential for the development of personalized and predictive medicine of the future.