The objective of the present work was to investigate the effect of formulation excipients on human stratum corneum (SC) barrier function in vivo. Two formulations, an ointment and an oil-in-water cream, were applied to the skin of human volunteers under both occlusive and non-occlusive conditions. The effects of each treatment were then evaluated using three non-invasive biophysical techniques: transepidermal water loss (TEWL), impedance spectroscopy (IS) and attenuated-total-reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. These measurements were combined with a simple tape-stripping protocol to allow information to be derived across the entire SC. IS and TEWL provided basic information on the effect of each formulation on skin barrier function, while ATR-FTIR enabled (i) the tracking of formulation excipients and evaluation of their concentration profiles within the SC, and (ii) deduction of mechanistic detail with which to explain the TEWL and IS results. It was found that occlusion of the skin either in the presence or absence of the cream caused TEWL to be increased when the treatment was terminated at 6 h. Uptake of ointment into the SC, on the other hand, inhibited the post-application TEWL rate. In parallel, treatment with the ointment caused an increase in relative low-frequency skin impedance, consistent with the entry of additional lipophilic constituents into the SC. The latter was confirmed by ATR-FTIR spectroscopic measurements. Overall, the combined use of the three biophysical measurements allowed formulation effects on, and uptake into, the SC to be deduced and evaluated, and the approach may prove useful for the future selection and optimization of topical drug delivery vehicles.