During the proteomic era, one of the most rapidly growing areas in biomedical research is biomarker discovery, particularly using proteomic technologies. Urinary proteomics has become one of the most attractive subdisciplines in clinical proteomics, as the urine is an ideal source for the discovery of noninvasive biomarkers for human diseases. However, there are several barriers to the success of the field and urinary proteome analysis is not a simple task because the urine has low protein concentration, high levels of salts or other interfering compounds, and more importantly, high degree of variations (both intra-individual and inter-individual variabilities). This article provides step-by-step practical points to perform urinary proteome analysis, covering detailed information for study design, sample collection, sample storage, sample preparation, proteomic analysis, and data interpretation. The discussion herein should stimulate further discussion and refinement to develop guidelines and standardizations for urinary proteome study.