The transdermal route offers advantages for delivery of peptides and proteins. However, these polar and large molecules do not permeate the skin barrier well. Various enhancement methods have been employed to address this problem. Iontophoresis is one of the methods that shows promise but its application to peptide delivery has yet to be fully explored. This study investigates the effects of different molecular properties and iontophoretic conditions on the skin permeation of peptides. In this study, the permeation of alanine-tryptophan dipeptide (MW 276 Da), alanine-alanine-proline-valine tetrapeptide (MW 355 Da), Argireline® (Acetyl hexapeptide-3, MW 889 Da) and Triptorelin acetate (decapeptide, MW 1311 Da) through excised human skin under passive or iontophoretic current of 0.4 mA was investigated. The effects of pH change (3.0-7.4, to provide different net negative, neutral, and positive charges) to the peptide, donor concentration (1-10 mg/ml), background electrolyte (34-137 mM NaCl and/or 5-20 mM HEPES) and current direction (anodal vs cathodal) were also studied. Peptides were analysed by high-performance liquid chromatography or liquid scintillation counting. Iontophoresis led up to a 30 times increase in peptide permeation relative to passive permeation for the peptides. Electroosmosis was an important determinant of the total flux for the high molecular weight charged peptides. Electrorepulsion was found to be considerable for low molecular weight charged moieties. Permeation was decreased at lower pH, possibly due to decreased electroosmosis. Results also showed that 10 times increase in donor peptide concentration increases permeation of peptides by about 2-4 times and decreases iontophoretic permeability coefficients by about 2.5-5 times. The addition of extra background electrolyte decreased the iontophoretic permeation coefficient of peptides by 2-60 times. This study shows that iontophoretic permeation of peptides is affected by a number of parameters that can be optimized for effective transdermal peptide delivery.