The differentiation of BK virus-associated renal allograft nephropathy (BKVAN) from acute allograft rejection (AR) in renal transplant recipients is an important clinical problem because the treatment can be diametrically opposite for the two conditions. The aim of this discovery-phase biomarker development study was to examine feasibility of developing a noninvasive method to differentiate BKVAN from AR. Surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry analysis was used to compare proteomic profiles of urine samples of 21 patients with BKVAN, 28 patients with AR (Banff Ia to IIb), and 29 patients with stable graft function. SELDI analysis showed proteomic profiles that were significantly different in the BKVAN group versus the AR and stable transplant groups. Peaks that corresponded to m/z values of 5.872, 11.311, 11.929, 12.727, and 13.349 kD were significantly higher in patients with BKVAN. Bioinformatics analyses allowed distinction of profiles of patients with BKVAN from patients with AR and stable patients. SELDI profiles also showed a high degree of reproducibility. Proteomic analysis of urine may offer a noninvasive way to differentiate BKVAN from AR in clinical practice. The identification of individual proteomic peaks can improve further the clinical utility of this screening method.