The aim of this work was to study the in vitro factors affecting transdermal iontophoretic delivery of methotrexate across hairless rat skin. Initial screening studies evaluated the effect of ionic strength and donor concentration. A response surface model using factorial design shows an increase in the cumulative amount of methotrexate delivered (Y1) with an increase in current density (X1) and time of application (X2). However, 10 min iontophoresis and 0.05 mA/cm2 current density did not show an increase in delivery with an increase in current density or time of application, respectively. The factorial design was able to identify the optimal parameters that would have been difficult to predict with a conventional one at a time-experimental approach.