Clinical management of prostate cancer remains a significant challenge due to the lack of available tests for guiding treatment decisions. The blood Prostate-Specific Antigen (PSA) test has facilitated early detection and intervention of prostate cancer. However, blood PSA levels are less effective in distinguishing aggressive from indolent prostate cancers and other benign prostatic diseases. Thus, the development of novel approaches specific for prostate cancer that can differentiate aggressive from indolent disease remains an urgent medical need. In the current study, we evaluated urine specimens from prostate cancer patients instead of serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the aim of identifying effective prostate cancer biomarkers. Glycoproteins from urine samples of prostate cancer patients with different Gleason scores were characterized via solid phase extraction of N-linked glycosite-containing peptides and LC-MS/MS. In total, 2923 unique glycosite-containing peptides were identified. Comparison of urine-based glycoproteins with those identified from aggressive and non-aggressive prostate cancer tissues as well as sera from prostate cancer patients revealed that the majority of aggressive prostate cancer-associated glycoproteins were more readily detected in patient urine than serum samples. Our data collectively indicate that urine provides a highly reliable source for biomarker testing in patients with aggressive prostate cancer.