In this review, we aim to give a concise and selective overview of noninvasive biophysical analysis techniques for skin barrier analysis (transepidermal water loss, electrical methods, confocal Raman microspectroscopy, sebumeter, reflectance spectrophotometry, tristimulus colorimetry, diffuse reflectance spectroscopy and reflectance confocal microscopy), including advantages and limitations. Rather than giving an exhaustive description of the many techniques currently available, we show the usefulness of a representative selection of techniques in the functional and morphological evaluation of the skin barrier. Furthermore, we introduce human minimally invasive skin challenging models as a means to study the mechanisms regulating skin homoeostasis and disease and subsequently show how biophysical analysis techniques can be combined with these in vivo skin challenging models in the functional and morphological evaluation of the skin barrier in healthy human skin. We are convinced that the widespread application of biophysical analysis techniques in dermatological practice and in cosmetic sciences will prove invaluable in offering personalized and noninvasive skin treatment solutions. Furthermore, combining the human in vivo challenging models with these novel noninvasive techniques will provide valuable methodology and tools for detailed characterization of the skin barrier in health and disease.