Abnormal methylation of urinary TWIST1 and NID2 conferred high sensitivity and specificity for the detection of urothelial carcinoma.We examine the performance of the urine-based TWIST1/NID2 methylation assay with the addition of urine cytology for the detection of urothelial carcinoma.A prospective multi-institutional study was conducted to assess the performance of a methylation assay for patients with hematuria or under surveillance for non-muscle invasive bladder cancer (NMIBC). All patients underwent cystoscopy, a methylation assay, and cytology. Receiver operator characteristic (ROC) curves were constructed for cytology alone, the methylation assay alone, and a combined model. Areas under the curve (AUC) were compared using likelihood ratio tests.A total of 172 patients were enrolled (37% for hematuria and 63% NMIBC). The AUC for cytology alone with equivocal cytologies positive was 0.704, and improved to 0.773 with the addition of the DNA methylation assay (p < 0.001). When the equivocal cytologies were considered negative, the AUC improved from 0.558 to 0.697 with the addition of the DNA methylation assay (p = 0.003).Addition of a TWIST1/NID2-based DNA methylation assay adds diagnostic value to urine cytology and the model is sensitive to the classification of equivocal cytology.