As noninvasive and easily available biological fluid, urine is becoming an ideal sample for proteomic study. In recent years, researchers endeavored in profiling urinary proteome and discovering potential disease biomarkers. However, there are still many challenges in the studies of urinary proteome for the complexity of urine. In this article, we review current status of urinary sample preparation, including collection, storage, and extraction of urinary proteins, and the overall urinary proteome analysis so far, which may be helpful for urinary proteome analysis.