The urothelium plays a critical role in the bladder as a permeability barrier to urine. Whereas it was once considered a simple physical barrier, it is increasingly evident that urothelium has a regulatory role in maintaining the barrier both through self-repair and by mediating the transport of ions and small molecules across the transcellular and paracellular interfaces. The development of cell culture systems that replicate the morphological and differentiated features of human urothelium provides a versatile in vitro tool for exploring molecular and functional relationships in normal bladder physiology and for examining inherent changes in the urothelia of patients with dysfunctional bladder syndromes. In addition, it provides a useful platform to study the effect of pharmacological treatment on urothelial barrier function. In this review, we describe the development of differentiated urothelial cell constructs from in vitro-propagated normal human urothelial cells, and the application of methods to assess barrier function using transepithelial electrical resistance, water, urea, and dextran transport as objective and quantifiable parameters.