Urine-derived stem cells (USCs) are multipotent stem cells obtained from human urine, offering a noninvasive and accessible source for both autologous and allogeneic therapies for multiple conditions including acute kidney injury (AKI) due to their renal origin. In our previous study, USCs were tracked in mouse models of AKI including rhabdomyolysis induced by glycerol injection. To track their migration in vivo, 1 × 106 luciferase-labeled USCs (luc-USCs) were administered to mice via intraperitoneal injection. In this chapter, we outline a comprehensive protocol for isolating and culturing USCs, as well as transfecting them with luciferase piggyBac transposon plasmids to confer expression of luciferase for tracking. We also detail the use of quantitative bioluminescence tomographic imaging (qBLT) for tracking USC migration and biodistribution, providing accurate spatial and temporal insights. We describe the procedure for generating 3D bioluminescent images and analyzing the data using InVivoAX™ software, offering a precise methodology for studying cell localization in animal models.