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Bladder urothelial carcinoma (BUC) is the most lethal malignancy of the urinary tract. Treatment for the disease highly depends on the invasiveness of cancer cells. Therefore, a predictive biomarker needs to be identified for invasive BUC. In this study, we employed proteomics methods on urine liquid-based cytology (LBC) samples and a BUC cell line library to determine a novel predictive biomarker for invasive BUC. Furthermore, an in vitro three-dimensional (3D) invasion study for biological significance and diagnostic validation through immunocytochemistry (ICC) were also performed. The proteomic analysis suggested moesin (MSN) as a potential biomarker to predict the invasiveness of BUC. The in vitro 3D invasion study showed that inhibition of MSN significantly decreased invasiveness in BUC cell lines. Further validation using ICC ultimately confirmed moesin (MSN) as a potential biomarker to predict the invasiveness of BUC (p = 0.023). In conclusion, we suggest moesin as a potential diagnostic marker for early detection of BUC with invasion in LBC and as a potential therapeutic target.