For the development of novel strategies for drug administrations, extensive knowledge over the delivery pathways, the physicochemical properties of the involved biological barriers, and the interactions with the drug are of paramount importance. In this thesis, I employed biophysical techniques like time-resolved fluorescence spectroscopy and microscopy in conjunction with advanced analytical tools to gain new insight into the structure of epithelial barriers as well as the biomolecular interactions of drugs and drug mimetics in the tissue. Fluorescent molecular rotors are fluorophores, which can be used to sense local nanoviscosity using their fluorescence lifetime. I applied these probes to artificial vesicles, in order to determine membrane viscosities and lipid phase transitions. In living cells of the gastrointestinal tract, they were used to map the 3D viscosity profile of the mucus layer lining the small intestine and characterize this diffusion barrier with unprecedented precision and spatial resolution. The extracted gradients and heterogeneities of the mucin network were compared based on culturing circumstances of the mucus producing epithelial cells, setting a framework for physiological cell growing conditions. In fluorescence lifetime imaging microscopy (FLIM), the target fluorescence of a molecular of interest can be discriminated from the autofluorescent background of the surrounding tissue based on the fluorescence lifetime. I used this advantage to generate background free penetration profiles of the drug mimetic Nile Red in human skin to extract biophysical properties of the outermost barrier of the body and compare unaided penetration with nanoparticle-assisted administration through various transdermal delivery routes. We finally developed a novel platform for labeling biomolecules with a fluorophore and a spin label. The multiplexed read-out of FLIM and EPR data facilitates the simultaneous determination of spatially resolved target molecule concentration and characterization of its immediate environment while avoiding analytical inconsistencies resulting from individual labeling. This thesis demonstrates the capabilities of time-resolved fluorescence techniques when paired with proper analytical strategies for the application in biophysical research and the development of new drug delivery systems.