Nucleocytoplasmic transport is crucial not only for basic cellular activities but also for the physiological adaptation of cells to various environmental stimuli that affect development, cell-fate determination, or disease development. The basic transport mechanisms have been revealed during the past two decades through the identification and biochemical characterizations of factors mediating the transport, dissecting the transport process and examining the function of nuclear pore complexes (NPCs). In this chapter, we describe methods for a nuclear transport reconstitution assay using digitonin-permeabilized mammalian cells. The transport assay can be generally conducted in the lab without special equipment. The assay system is efficient and significantly contributes to the study of nucleocytoplasmic transport.