Urothelial cells contribute to bladder functions, including urine storage, urine emptying, and innate immune response. Functional studies of urothelial cells usually use either freshly isolated cells or cultured cells. Most methods of isolating urothelial cells require enzymes; however, these techniques remove proteins that connect the cells and disrupt the orientation of the cells within the multilayered urothelium. In addition, PCR or immunoblot results obtained from homogenates of bladder mucosa or whole bladder do not represent pure urothelial cells. We describe a dissection process that does not require enzymes and is able to obtain pure urothelial tissues from mice and humans. This method can isolate single urothelial cells for electrophysiology in situ and can also isolate pure urothelial tissue for PCR, microarray, and immunoblot procedures. The time required to obtain urothelial tissue from one mouse bladder is 15–20 min. This method is simple and time efficient as compared with alternative methods and therefore facilitates our understanding of urothelial biology.