We describe here a convenient procedure for assessing the relative cell permeability of chemical compounds. The assay can be used in a high-throughput mode and is particularly applicable for the evaluation of the relative permeability of compounds in a combinatorial library. The compound of interest is conjugated to a dexamethasone derivative. The entry of the conjugate into living mammalian cells triggers the nuclear transport of a Gal4 DNA binding domain–glucocorticoid receptor ligand binding domain–VP16 activation domain fusion protein and, consequently, the activation of a Gal4-responsive luciferase reporter gene. The relative cell permeability is thus described quantitatively by the level of luciferase expression. The experiments take only a few days once chemical synthesis and conjugation are finished.