Dogs share many anatomical and physiological characteristics with humans and, in certain instances, represent a more reliable research model than rodents for some diseases, including several types of human cancer. Advances in adult stem cell research have accelerated the development of 3D organoid cultures in non-model animals, such as dogs. These advancements have the potential to significantly impact disease modeling, biomarker discovery, and drug development for both human and canine diseases, including bladder cancer (BC). In this Chapter, we present a standardized protocol for generating BC organoids from canines. The chapter describes the detailed methodology for establishing canine BC organoids from urine or tissue-derived stem cells. Additionally, it highlights the potential of these organoids to recapitulate the expression of key markers from their original tissues and their utility in various biomedical applications such as drug screening, gene mutation analysis, and toxicology. Finally, we discuss potential future research directions for canine BC organoids in translational research.