Abstract Purpose: To investigate the use of plasma and urine DNA mutation analysis for predicting neoadjuvant chemotherapy (NAC) response and oncological outcome in patients with muscle-invasive bladder cancer. Experimental Design: Whole-exome sequencing of tumor and germline DNA was performed for 92 patients treated with NAC followed by radical cystectomy (RC). A custom NGS-panel capturing approximately 50 mutations per patient was designed and used to track mutated tumor DNA in plasma and urine. A total of 447 plasma samples, 281 urine supernatants, and 123 urine pellets collected before, during, and after treatment were analyzed. Patients were enrolled from 2013 to 2019, with a median follow-up time of 41.3 months after RC. Results: We identified tumor DNA before NAC in 89% of urine supernatants, 85% of urine pellets, and 43% of plasma samples. Tumor DNA levels were higher in urine supernatants and urine pellets compared with plasma samples (P < 0.001). In plasma, detection of circulating tumor DNA (ctDNA) before NAC was associated with a lower NAC response rate (P < 0.001). Detection of tumor DNA after NAC was associated with lower response rates in plasma, urine supernatant, and urine pellet (P < 0.001, P = 0.03, P = 0.002). Tumor DNA dynamics during NAC was predictive of NAC response and outcome in urine supernatant and plasma (P = 0.006 and P = 0.002). A combined measure from plasma and urine supernatant tumor DNA dynamics stratified patients by outcome (P = 0.003). Conclusions: Analysis of tumor DNA in plasma and urine samples both separately and combined has a potential to predict treatment response and outcome.