Since its introduction in 1998, the clinical indications of microneedle therapy system (MTS) were ever enlarging. One of the most important applications of MTS is to improve the efficacy of transdermal drug delivery. Although widely adopted in medical practice, the most effective strategy employing MTS was not fully evaluated. In this study, the time-course of the skin penetration of lipophilic dye rhodamine as model drug was investigated, and different application sequences of the microneedle roller and drug fromulation, i.e. roller-first vs. drug-first vs. drug-roller-drug, were compared in terms of penetration efficacies. The skin penetration of lipophilic dye rhodamine on porcine skin was evaluated by the fluorescent image analysis. The time-course of transdermal penetration of lipophilic dye rhodamine showed interesting chronological pattern: initial rapid penetration, followed by short-term plateau, then second rapid increase in penetration, and final plateau. With respect to the efficacy of penetration, drug-roller-drug method was superior to other application modalities. Result will be discussed the percutaneous penetration of drug can be 19 effectively enhanced by the repeated application of drug on the skin surface before and after microhole formation by microneedle roller. This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea. (A090180)