By exploiting the unique physicochemical properties of nanoparticles, novel sophisticated approaches in biomedical imaging, therapy and drug delivery have emerged, forming an innovative research field, coined Nanomedicine. In view of the constant quest for personal medicine and precise targeted therapeutics with minimized side effects, several of these approaches have the potential to revolutionize the medical field. Despite our current efficient intellectual framework about nanoparticle behavior in biological systems, more and more gaps in our knowledge appear, if we question the ultimate in vivo fate of nanoparticles after administration. The unraveling of the pharmacokinetics and pharmacodynamics, in other words, a comprehensive scientific understanding of the interactions between biological entities and nanomaterials, remains essential to assure a successful clinical translation. This results in a continuously growing demand for in vivo and in situ investigation methods, which are applicable in realistic biological environments, in order to assess and predict the performance and safety of nanodrugs in an early development stage. In this thesis, the in situ nanoparticle behavior in biological environments was studied by means of light scattering. Herein, the feasibility of employing X-rays to study nanoparticles in dense biological systems is explored. In a series of in situ X-ray studies, the following aspects were addressed: (1) Assessing the performance of engineered polymer-based nanoparticle coatings in presence of proteins (2) studying the interactions between nanoparticles and biological entities (3) investigating the dynamic profiles and structural alterations of nanoparticles and (4) monitoring the degradation behavior of hybrid nanostructures for drug delivery applications.