The large volume and diversified nanomedicine market, undergoing a rapid growth, relies not only on the creation and applicative exploration of nanocarrier-based medicines showing significant potential, but in particular, demands a quantitative assessment of their physicochemical properties. In this study, we demonstrate the in situ assessment of multifunctional biodegradable nanoparticle (NP) entries as core components of nanoscale drug delivery systems (NDDSs) by making use of analytical ultracentrifugation (AUC). We determine and elucidate the following characteristics of NPs in NDDSs: NP density and size, targeting dye functionality, encapsulated and free drug, surfactant, and also NP drug release dynamics, quantitatively interconnected to NP degradation. In concept, we demonstrate this by multidetection AUC experiments at variable speed and time profiles. We could verify the quantitative and accurate nature of AUC for assessment of NDDSs, that is, also future nanomedicines. This concerns modeled and real life solution application formats such as cell culture media and human serum.