Simple SummaryAccurate, non-invasive methods that can diagnose bladder cancer (BC) are needed to detect BC in its early stages and reduce the burden on patients with hematuria. We developed uAL100, a method that accurately diagnoses BC by identifying tumor DNA in urine samples using targeted deep sequencing. We demonstrate that uAL100 accurately detects putative BC driver mutations in the urine without information about the tumor.AbstractPatients with hematuria are commonly given an invasive cystoscopy test to detect bladder cancer (BC). To avoid the risks associated with cystoscopy, several urine-based methods for BC detection have been developed, the most prominent of which is the deep sequencing of urine DNA. However, the current methods for urine-based BC detection have significant levels of false-positive signals. In this study, we report on uAL100, a method to precisely detect BC tumor DNA in the urine without tumor samples. Using urine samples from 43 patients with BC and 21 healthy donors, uAL100 detected BC with 83.7% sensitivity and 100% specificity. The mutations identified in the urine DNA by uAL100 for BC detection were highly associated with BC tumorigenesis and progression. We suggest that uAL100 has improved accuracy compared to other urine-based methods for early BC detection and can reduce unnecessary cystoscopy tests for patients with hematuria.