Background: the median overall survival (OS) in metastatic urothelial carcinoma (mUC) treated with multiagent therapy is approximately 13 months (mo). Around 69% of these tumors harbor potential therapeutic targets. Real-world evidence on using Next Generation Sequencing (NGS) in mUC management is limited. Methods: we conducted a single-center analysis on a real-world cohort of patients (pts) with mUC. The study population included pts aged >18 years with diagnosis of mUC studied by NGS at the moment of starting immunotherapy (enrollment period: 09/04/2019 – 03/25/2022). Median OS and median progression-free survival (PFS) were determined using Kaplan-Meier curves. Results: we included 43 pts. Median age at mUC diagnosis was 67 years; 38 (88.4%) pts were men; 38 (88.4%) pts were ECOG 0-1 at mUC diagnosis; one or more adverse prognostic factors (Bellmunt Risk Score) were identified in 33 (76.7%) pts; primary tumor site was lower tract in 36 (83.7%) pts. All pts were treated with platinum-based combinations (neoadyuvant, adyuvant or first line) and immunotherapy. Pathogenic alterations were found in 25 (58.1%) pts. In the group of 25 pts with pathogenic alterations, 17 pts progressed to immunotherapy and 7 pts of them received targeted therapies; 10 pts could not receive targeted therapies due to poor prognosis. In the group of 18 pts without pathogenic alterations, 11 pts progressed to immunotherapy and 8 pts of them received other chemotherapy options; 3 pts could not receive other chemotherapy options due to poor prognosis. The median OS (time since mUC diagnosis) among 25 pts with pathogenic alterations was 30.2 (CI 95%: 18.9-41.5) mo compared to 22.9 (CI 95%: 12-33.9) mo among 18 pts without pathogenic alterations, (p = 0.557). Focusing on the subgroup of 7 pts treated with targeted therapies (Table), we found an objective response rate (ORR) of 42.9%; the median PFS was 7.3 (CI 95%: 6.7-7.9) mo and the median OS (time since beginning targeted therapies) was 10.9 (CI 95%: 2.4-19.5) mo. Conclusions: We recommend that all pts with mUC undergo NGS at the time of diagnosis, given the high percent (58.1%) of pathogenic alterations in our real-world cohort and the efficacy in terms of ORR, PFS and OS in pts treated with targeted therapies.