Purpose: Tape-stripping the stratum corneum (SC) is used in the assessment of dermatopharmacokinetics (DPK). The amount of SC per tape can be determined gravimetrically, but a novel imaging method offers advantages in terms of sensitivity, reproducibility, precision, stability and speed. High-resolution images, acquired under controlled conditions, are analysed in terms of pixel greyscale values and distributions, and their usefulness in DPK studies is demonstrated in this study using acyclovir. Methods: At all tape-stripped sites, the SC amount per tape was measured gravimetrically and by imaging. In a first series of experiments, untreated sites were stripped to determine total SC thickness. Subsequently, post-application of two acyclovir creams, drug-permeation profiles were constructed. Results: The greyscale values from the imaging data can be used directly to estimate total SC thickness and DPK parameters. The results compared favourably with the traditional weighing method. The concentration of drug on each tape, as a function of the relative position within the SC, permitted diffusivity and partitioning parameters characterising the penetration of acyclovir to be derived. Conclusion: The new imaging approach offers a sensitive, reproducible, precise, and rapid technique to quantify the relative SC amount removed on tape-strips, and facilitates the acquisition of DPK data.