Today’s nanomedicine struggles with low success rates in clinical trials. Even though considerable efforts have been made, it is still not possible to exploit its full potential. One of the major problems is the clinical translations from pre-clinical in-vitro and ex-vivo studies to in-vivo applications. While this problem can be attributed to different reasons, such as the limited predictive power of biological test models, experimental methods, their analyses, and interpretations, the lack of information on the interactions between nanoparticles and the plethora of biomolecules poses the main challenge. In order to shed some light on these interactions, this thesis presents the development and application of fluorescence lifetime spectroscopy and imaging approaches for dendritic nanoparticle systems. By applying fluorescence lifetime probes, the scope of fluorescence read-outs will be extended to monitor biological nanoparticle interactions. Identifying interactions between nanoparticles and target structures and elucidating whether loaded drugs reach their biological targets remain important problems to be solved. The thesis aims to identify nanoparticle locations, interaction states, and molecular environments in diverse in-vitro and ex-vivo test systems by TCSPC-based time-resolved fluorescence experiments and novel analysis approaches. First, the sensitivity of fluorescence lifetime probes is characterized in model experiments on a molecular level. By means of a fluorescent molecular rotor (FMR) probe, molecular interactions between dendritic nanoparticles and biomolecules like serum albumin are investigated. They are responsible for the so-called protein corona formation, a crucial effect on the systemic application of nanomedicine as it alters further interactions of the nanoparticle during its transport through the body. Second, FMRs are applied to explore their potential for probing nanoparticles’ molecular conformational changes and drug release mechanisms. Moreover, I apply a cluster-based analysis tool for quantitative fluorescence lifetime imaging microscopy (FLIM) to living dermal cells interacting with cargo-loaded and FMR-equipped dendritic nanocarriers. Based on distinct changes in fluorescence lifetime parameters, I identify unknown cellular targets in primary human keratinocytes, quantify the binding affinity of a target surface receptor, visualize intracellular cargo release, and further endocytotic interactions. Thereby, the cellular uptake behavior and fate of the nanocarrier system was characterized in a time-resolved and non-invasive manner. In addition, I investigate the penetration behavior and identify target structures of dendritic nanocarriers after topical administration on different in-vitro and ex-vivo skin models by confocal FLIM. These experiments reveal specific interactions within the stratum corneal layers, the major interaction site of nanoparticles after topical application. Multiphoton excitation enables the live-tissue FLIM tomography of these interactions in reconstructed human tissue. This shows significant differences in the interaction modes of a dendritic nanocarrier with models of healthy skin and skin suffering from non-melanoma cancer.