Human urine is an attractive and informative biofluid for medical diagnosis, which has been shown to reflect the (patho)-physiology of not only the urogenital system, but also others such as the cardiovascular system. For this reason, many studies have concentrated on the study of the urine proteome, aiming to find relevant biomarkers that could be applied in a clinical setting. However, this goal can only be achieved after reliable quantitative and qualitative analysis of the urinary proteome. In the last two decades, MS-based platforms have evolved to become indispensable tools for biomarker research. In this review, we will present and compare two of the most clinically relevant analytical platforms that have been used for the study of the urinary proteome, namely CE-based ESI-MS and classical MALDI-MS. These platforms, although not directly comparable, have been extensively used in proteomic profiling and therefore their comparison is fundamentally relevant to this field.