Current topical nonsteroidal anti-inflammatory drugs (NSAIDs) showed marginal efficacy in treatment of musculoskeletal disorders due to their fast clearance by skin blood flow and thus little direct penetration into the underlying muscle and joint tissues. Using ketoprofen (Kt) as a model NSAID and converting it to a cationic ester prodrug ketoprofen choline chloride (KCC), this study was to investigate the iontophoresis delivery of the prodrug KCC for improving the drug retention in the skin and the direct penetration into underlying tissues. From in vitro flux study, anodal iontophoresis of KCC showed 5 times higher flux than cathodal iontophoresis of Kt across human epidermis skin, and also 1.5 times higher across full thickness rat skin. From in situ dual agar gel model rat study, anodal iontophoresis of KCC showed 35 times more drug penetrating across the live skin into underlying agar gel and 22 times more drug retained in the skin than those from cathodal iontophoresis of Kt. Co-iontophoresis of a vasoconstrictor phenylephrine with KCC did not show better result than the iontophoresis of KCC alone. Overall, iontophoresis delivery of the cationic prodrug KCC showed great potential for direct penetration into local tissues underneath the skin.