We have evaluated a transdermal delivery system containing prednisolone by in vitro/in vivo/in silico experiments. An in vitro study using hairless mouse skin showed that the steady-state rate of the drug penetration across the skin with polyoxyethylated oleyl ether (POE) and isopropyl myristate (IPM) as enhancer was 444-fold greater than that without enhancer. The plasma concentration after the application of transdermal prednisolone delivery system with POE and IPM was also measured using rats in vivo. The transdermal application showed an initial transient absorption process for 2 h after application and thereafter a constant concentration level of drugs was maintained for about 9 h. Moreover, the plasma concentration after the application of the transdermal prednisolone delivery system to the central abdominal skin of rats was simulated using the skin permeation parameters determined from the in vitro experiment. The simulated profiles agreed with the in vivo experiment.