Background: Despite recent therapeutic advancements for patients with mUC, including checkpoint inhibitors (CPIs), anti-FGFR and antibody-drug conjugates (ADCs), biomarker data to predict therapeutic response and identify mechanisms of resistance remain limited. Ongoing randomized clinical trials evaluating the combination of CPIs and ADCs in these pts underscore the need to develop biomarkers to better understand who might best respond to these therapies. Here, we employ a liquid biopsy approach to molecularly characterize circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), including those isolated with Trop-2, the target of the novel drug Sacituzumab Govitecan and Datopotamab Deruxtecan. Methods: 104 blood samples serially collected from pts being treated for mUC were used for CTC collection using anti-EpCAM and Trop-2 antibodies in parallel. CTCs were isolated and stained for immune markers PD-L1 and HLA I using the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. Plasma was isolated for paired analysis of CTC and ctDNA content. CTCs were analyzed for enumeration and single-cell protein analysis. Overall survival (OS) was defined from time of sample collection, and survival analyses were performed using the Kaplan-Meier method. Results: We observed significantly shorter OS in pts with higher CTC burden (≥ 20 CTCs/7.5 mL blood) in samples captured with either EpCAM (median 34.1 vs 3.1 mo, P < 0.01) or Trop-2 (median 34.1 vs 6.2 months, P < 0.01). Furthermore, EpCAM and Trop-2 CTC burdens were higher at progression timepoints relative to responding or stable timepoints, when stratified according to treatment modality (table). Inter and intra-patient heterogeneous PD-L1 and HLA I expression and co-expression was observed on CTCs although no significant differences were seen globally between EpCAM and Trop-2 CTCs. In a cohort of pts treated with CPIs, we observed dynamic phenotypic changes in the ratio of HLA:PD-L1 that differed between EpCAM and Trop-2 CTCs. ctDNA analysis identified concordant increases in tumor fraction and acquired genomic mutations. When examined longitudinally across individual pts, these findings in tandem with changes in enumeration, suggest the emergence of different CTC subpopulations that may serve as biomarkers of resistance. Conclusions: We identify the prognostic significance of CTC enumeration with parallel EpCAM and Trop-2 CTC isolation and demonstrate the utility of a liquid biopsy assay that can detect pharmacodynamic changes in CTC burden and immune marker expression throughout the course of treatment.