To increase the skin permeation of large peptides like insulin, it is necessary to utilize a combination of enhancement strategies. In this regard, this study investigated the effect of terpenes/EtOH combination in comparison to EtOH and neat terpene on transdermal iontophoretic permeation of insulin. Ex-vivo experiments were conducted using full thickness rat skin after pre-treatment for 2 h with 5% of menthol, menthone, cineole and pulegone in EtOH; EtOH alone; neat menthone with and without iontophoresis (0.5 mA/cm(2); 6 h). FT-IR studies were carried out using rat epidermal sheets after pre-treatment with enhancer solution for 2 h and tritiated water permeation studies was used to investigate the alteration in skin barrier property after enhancer or current treatment. The lag time was significantly reduced (P<0.05) with terpene/EtOH pre-treatment in comparison to passive control and EtOH pre-treatment, although there was no significant difference (P>0.05) among the terpenes. Synergistic enhancement in flux was observed with terpene/EtOH, and menthone/EtOH showed highest enhancement among the terpene/EtOH combinations. On the other hand, enhancement with neat menthone was higher than with menthone/EtOH. FT-IR studies showed that terpene/EtOH, EtOH and neat terpene act at the intercellular lipids. The skin barrier property was significantly (P<0.05) compromised with neat menthone treatment. Iontophoresis had a lesser effect on skin barrier property compared to chemical enhancer pre-treatment. Terpene/EtOH caused synergistic enhancement of insulin permeation when combined with iontophoresis and was influenced by the type and concentration of terpene.