Urine, as a noninvasive and easily available biological fluid, is increasingly considered an optimal specimen for the investigation of clinical disease biomarkers. In recent years, researchers have focused on profiling the urinary proteome and identifying potential biomarkers for diseases. However, investigating the urinary proteome faces challenges due to urine’s complexity. This chapter reviews urinary sample preparation, including urine collection, storage, and urinary protein extraction, and also analyzes the urinary proteome while discussing the influence of physiological factors on the normal human urinary proteome, which may advance research in this area.