Induced desquamation of urinary bladder epithelial cells, also called urothelial cells, is frequently used in studies of bladder epithelial regeneration and also in treating recurrent bacterial cystitis. Positively charged polymer chitosan is known to cause large-scale desquamation of terminally differentiated urothelial cells called umbrella cells. Aiming to compare the desquamation ability of another polycation poly-L-lysine, we studied the effect of this polymer on the functional and structural integrity of the urothelium in ex vivo and in vivo experiments. The urothelium was analyzed by measuring transepithelial electrical resistance, and the structural changes of its luminal surface were analyzed with scanning electron microscopy. The results revealed a selective and concentration-dependent desquamation effect of poly-L-lysine on superficial urothelial cells followed by quick regeneration of the urothelium, which functionally and structurally recovers in 2 to 3 h after poly-L-lysine-induced injury. Poly-L-lysine was thus proven to be a promising polymer to be used when desquamation of urothelial cells is required in basic and potentially clinical studies.