A novel application of high-speed two-photon microscopy was utilized to determine the optimum number of skin sites required to accurately determine the changes in transdermal transport properties incurred globally, over a clinically relevant area of skin. In contrast to the four to six skin sites (100 microm by 100 mirom area per site) examined previously, this study accounted for the fluorescent probe distributions at 400 consecutive skin sites, covering a total skin area of 2 mm by 2 mm. The oleic-acid-induced changes in the transdermal transport properties of the model hydrophobic probe, rhodamine B hexyl ester, and of the model hydrophilic probe, sulforhodamine B, for this 400-skin-site study exhibited different dependencies on sample size for each probe. Whereas the examination of six skin sites captures the relative changes in the global transdermal transport properties of the hydrophobic probe, the valid assessment of these changes for the hydrophilic probe requires a significantly larger sample size of at least 24 skin sites.