The objective of the present work was to investigate the effects of 3 combinatorial techniques (microneedle (MN), electroporation (EP), and sonophoresis (SN)) on the in vitro skin permeation of the hydrophilic macromolecular compound fluorescein isothiocyanate-dextran (FD-4; molecular weight (MW) 4.4 kDa). Assessment of the in vitro skin permeation of FD-4 was performed in porcine skin. MN, EP, and SN were used as physical enhancement methods, given the potential of their various mechanisms. The total cumulative amount of FD-4 that permeated through treated skin using 2 or 3 combined methods, i.e. , MN+EP, MN+SN, EP+SN, and MN+EP+SN, was investigated. Microconduits created by MN alone and in combination with the other techniques were observed under confocal laser scanning microscopy (CLSM). The histology of the treated skin was examined. In vitro skin permeation experiments revealed that the total cumulative amount of FD-4 that permeated porcine skin using 3 combined techniques (MN+EP+SN) was greater than the amount observed using a single method or 2 combinations (MN+EP, MN+SN, SN+EP). The histological images indicate no noticeable damage in the skin treated with all of the enhancement methods. These results suggest that MN+EP+SN may serve as a potentially effective combination strategy to transdermally deliver various hydrophilic macromolecules without causing structural alterations or skin damage.