Objective To provide preliminary clinical performance evaluation of a novel prostate cancer (CaP) assay, prostate-specific antigen/solvent interaction analysis (PSA/SIA) that focused on changes to the structure of PSA. Methods Two-hundred twenty-two men undergoing prostate biopsy for accepted clinical criteria at 3 sites (University Hospitals Case Medical Center in Cleveland, Cleveland Clinic, and Veterans Administration Boston Healthcare System) were enrolled in institutional review board–approved study. Before transrectal ultrasound–guided biopsy, patients received digital rectal examination with systematic prostate massage followed by collection of urine. The PSA/SIA assay determined the relative partitioning of heterogeneous PSA isoform populations in urine between 2 aqueous phases. A structural index, K, whose numerical value is defined as the ratio of the concentration of all PSA isoforms, was determined by total PSA enzyme-linked immunosorbent assay and used to set a diagnostic threshold for CaP. Performance was assessed using receiver operating characteristic (ROC) analysis with biopsy as the gold standard. Results Biopsies were pathologically classified as case (malignant, n = 100) or control (benign, n = 122). ROC performance demonstrated area under the curve = 0.90 for PSA/SIA and 0.58 for serum total PSA. At a cutoff value of k = 1.73, PSA/SIA displayed sensitivity = 100%, specificity = 80.3%, positive predictive value = 80.6%, and negative predictive value = 100%. No attempt was made in this preliminary study to further control patient population or selection criteria for biopsy, nor did we analytically investigate the type of structural differences in PSA that led to changes in k value. Conclusion PSA/SIA provides ratiometric information independently of PSA concentration. In this preliminary study, analysis of the overall structurally heterogeneous PSA isoform population using the SIA assay showed promising results to be further evaluated in future studies.