Urine testing has been used as a noninvasive, economical tool for disease screening, diagnosis, and surveillance. With the progress of urine detection technology, modern analysis platforms have effectively measured urinary proteomics, which can be used better to understand the pathophysiology and clinical translation of cancer. At present, urine biomarkers have been used to detect a variety of cancers, such as gastric cancer, pancreatic cancer, and glioma. This chapter mainly introduces the research progress of urine protein biomarkers based on tumor-bearing animal models and the clinical application of urine proteomics in cancer biomarker discovery.