The limitation of prostate specific antigen (PSA) for prostate cancer (PC) diagnosis is well-recognized. The Gleason score (GS) has been the most widely used grading system for prostate tumor differentiation and represents the best-established prognostic indicator for prostate cancer progression. However, a rapid and sensitive noninvasive diagnostic marker that differentiates GS-based prostate cancer disease progression is needed. As PC is becoming a leading cause of cancer related death for men in the U.S. and worldwide, an immediate need exists for an improved, sensitive, noninvasive, and rapid diagnostic test for PC screening. Here, we employed paper spray ionization-mass spectrometry (PSI MS)-based global metabolomics of urine liquid biopsies to distinguish between healthy (negative for any prostate specific health problems) and progressive PC states (low grade PC such as GS6 and high-grade PC such as GS7, GS8, and GS9). For PSI-MS-based direct untargeted metabolic investigation, a raw urine sample was directly pipetted onto a triangular paper substrate, without any additional sample preparation. Multivariate statistical analysis revealed distinct GS-specific metabolic signatures compared to a healthy control. Variable importance in projection from partial least-squares-discriminant analysis showed distinct metabolic patterns that were correlatively elevated with progressive disease and could serve as biomarkers for diagnosis of prostate cancer risk categorization.