Multiple targeted therapeutics have been approved by the FDA for mUC, including immune checkpoint inhibitors (ICIs) and more recently targeted agents for both FGFR and Nectin-4.FGFR3-aberrant and Nectin-4 expressing cells have been associated with an immunosuppressed phenotype. Given that less than half of all patients respond to these agents as monotherapies and less than 20% are eligible to receive salvage therapy, effective personalized treatment plans are critical. Typical biomarkers for ICIs such as PD-L1 and TMB have not been definitive in mUC, yet a biomarker-driven optimization of first-line therapy and subsequent sequencing have the potential to achieve higher and more durable response rates. The IO score is a 27-gene tumor immune microenvironment (TIME) classifier that has been associated with the clinical benefits of ICIs in multiple cancer types, including mUC. This study demonstrates that the IO score was associated with both progression-free survival (PFS) and overall survival (OS) in a real-world cohort of mUC patients treated with ICIs. Furthermore, the IO score was independent of and provided information incremental to TMB. Interestingly, the IO score predicted benefit in patients with high FGFR expression, despite conflicting data regarding response rates among the FGFR aberrant population. Taken together, these results demonstrate that the IO score assessment of the TIME is associated with a clinical benefit from ICI therapy and that this novel biomarker may inform therapeutic sequencing decisions in mUC, potentially improving outcomes for this notoriously difficult-to-treat disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-023-03401-x.