Immune checkpoint inhibitors (ICIs) are commonly used to treat patients with advanced urothelial cancer. However, a significant number of patients do not respond to ICI, and the lack of validated predictive biomarkers impedes the success of the ICI strategy alone or in combination with chemotherapy or targeted therapies. In addition, some patients experience potentially severe adverse events with limited clinical benefit. Therefore, identifying biomarkers of response to ICI is crucial to guide treatment decisions. The most evaluated biomarkers to date are programmed death ligand 1 expression, microsatellite instability/defective mismatch repair phenotype, and tumor mutational burden. Other emerging biomarkers, such as circulating tumor DNA and microbiota, require evaluation in clinical trials. This review aims to examine these biomarkers for ICI response in urothelial cancer and assess their analytical and clinical validation.