Transdermal iontophoresis offers an in vivo alternative to the strain-gauge model for measurement of vascular function but is limited due to lack of technical solutions for outcome assessment. The aims of this study were to, after measurement by polarized reflectance spectroscopy (PRS), use pharmacodynamic dose-response analysis on responses to different concentrations of acetylcholine (ACh); and to examine the effect of three consecutively administered iontophoretic current pulses. The vascular responses in 15 healthy volunteers to iontophorised ACh (5 concentrations, range 0.0001% to 1%, three consecutive pulses of 0.02 mA for 10 min each) were recorded using PRS. Data were fitted to a four-parameter logistic dose response model and compared. Vascular responses were quantifiable by PRS. Similar pharmacodynamic dose response curves could be generated irrespectively of the ACh concentration. Linearly increasing maximum vasodilatory responses were registered with increasing concentration of ACh. A limited linear dose effect of the concentration of ACh was seen between pulses. Polarized reflectance spectroscopy is well suited for measuring vascular responses to iontophoretically administrated ACh. The results of this study support further development of iontophoresis as a method to study vascular function and pharmacological responses in vivo.