Background: Given that ICIs benefit a minority of mUC patients and are associated with significant costs, biomarkers are necessary to optimally utilize them in the clinic. Although FOXP3+ T-cells have been associated with an immune-cold environment in many cancers, studies in urothelial carcinoma have shown an opposite trend. Methods: Formalin-fixed paraffin-embedded slides from tumor specimens were collected for patients with mUC treated with ICI at Dana-Farber Cancer Institute. A novel multiplex immunofluorescence (mIF) panel, ImmunoProfile, was performed for PD-L1, PD-1, FOXP3, CD8 and DAPI, then scanned by a Vectra Polaris platform. Regions of interest were defined and used for quantitative analysis using PerkinElmer/Akoya. Intratumoral (IT) and tumor-stroma interface (TSI) density (cells/mm²) of each cell type was calculated. Clinical data was collected through chart review, and associations between cell density and response were assessed. Wilcoxon Rank-Sum test between responders (CR/PR) and non-responders (SD/PD) was used to generate p-values, followed by Benjamini-Hochberg correction. Receiver-operating curve (ROC) and area-under-curve (AUC) calculations were performed to determine the optimal cutoff (OC) differentiating responders from non-responders. Cox proportional hazards models were used to estimate OS and PFS, accounting for type of therapy (single vs. combination ICI), baseline neutrophil-to-lymphocyte ratio (NLR), PD-L1 CPS, prior therapy, non-urothelial component, ECOG-PS and liver metastases (mets). Results: Of 35 patients assessed by ImmunoProfile, 32 were evaluable for response. Most patients (88%) were male and the median age at ICI start was 73 years. Median number of prior lines of therapy was 1 (range 0 – 3), and the majority (72%) was treated with single-agent ICI. Eight patients (25%) had CR/PR, eight had SD and 16 had PD as best response. Of all IF stains assessed, IT-TSI FOXP3 was the strongest predictor of objective response (q-value = 0.006), followed by IT-CD8 (q = 0.014). ROC analysis yielded an AUC of 0.812 (0.656 – 0.969) and the optimal cutpoint was set at 75 IT-TSI-FOXP3 cells/mm². ORR was 46% in FOXP3-high ( > 75/mm²) vs 14% in FOXP3-low mUCs. A combined model using IT-TSI-FOXP3 and clinical covariates (NLR, ECOG-PS, line of therapy, histology, and liver mets) had an AUC of 0.929. PFS was significantly longer in the FOXP3-high group (7.9 [5.7 – NR] months) compared to the FOXP3-low group (2.3 [2.1 – 6.1] months) on multivariable analysis (p = 0.007). OS also showed the same trend (p = 0.1). Conclusions: In this pilot study of ImmunoProfile, a novel mIF panel, higher FOXP3+ infiltration in tumors was associated with better outcomes and a composite clinico-IHC panel exhibited robust prognostic impact in mUC pts treated with ICI. Further study of this mIF panel is warranted to implement it in routine practice.