Gap in Knowledge: No accurate, non-invasive tests are currently available to rule in patients with hematuria or a history of bladder cancer (BCa) who need an invasive cystoscopy to evaluate the presence of a bladder tumor. Background: The most common presenting symptom in patients with BCa is hematuria. In two large studies, ~11% of patients with hematuria were noted to harbor BCa. Published guidelines recommend these patients to obtain voided urinary cytology (VUC), in addition to cystoscopic evaluation. Cystoscopy is an invasive, uncomfortable and expensive procedure associated with side effects such as transient voiding symptoms, hematuria, UTI, and stenosis of the urethra, whereas, VUC has limited sensitivity of 25-40% in detecting BCa (specificity is >90%), especially for low-grade and low-stage tumors. While some commercially available urine-based assays for the detection of BCa are available, many suffer from a reduction in assay specificity compared to VUC (e.g., NMP-22 and BTA). Furthermore, as single markers, these assays, including VUC have insufficient predictive power to be applied to the management of individual patients, and importantly, these techniques are complex, and require skillful interpretation. Our current NIH/NCI R01 application is testing a multiplex electrochemoluminescent (MEC) immunoassay’s ability to detect our BCa- associated diagnostic signature in voided urine samples from subjects with gross hematuria and subjects with a history of BCa on tumor surveillance. Since the inception of the R01 application, we have developed and validated a multiplex bead-based (MBB) immunoassay, which possesses improved operational characteristics such as area under receiver operating characteristic, sensitivity and specificity (0.942: CI 0.8645 – 0.9627, 93% and 95%, respectively using MBB platform vs. 0.892: CI 0.850 - 0.934, 85% and 81%, respectively using MEC platform). Because of the substantial improvement in the MBB assay, we seek to incorporate the evaluation of the MBB assay into the current R01 grant, and therefore compare the MBB assay to the MEC assay in this supplemental application. Hypothesis: The MBB assay is more sensitive and specific than the MEC assay in detecting our BCa-associated diagnostic signature in voided urine samples. Methodology: As with the MEC assay, we will perform the MBB assay in a CLIA-certified laboratory. We will then compare and contrast the operational characteristics of the MBB and MEC assays in the current, large multi-center prospective studies. Upon completion of the proposed work, we expect to show that the MBB assay is non- inferior to the MEC assay in detecting our BCa signature. Therefore, MBB assay will be transitioned into the laboratory and used for our subsequent studies with the ultimate goal of accelerating the pace of translation of our NCI-supported methods/assays/technologies to the clinic, which is the premise of PAR-17-003.