Iontophoresis technology could improve the efficiency of transdermal drug delivery through the skin and be a promising prospective tool for clinical drug therapy in the near future. Surface-enhanced Raman spectroscopy (SERS) was used to analyze the concentration distribution of penicillin sodium in the skin of a mice model, and the iontophoresis delivery efficiency of drug percutaneous permeation was evaluated with various times and concentrations of penicillin sodium through Raman spectra. The results showed both the action time and drug concentration for iontophoresis can deeply influence transdermal drug delivery effects, and the Raman spectrum might be an effective method to evaluate transdermal drug delivery efficiency.