Diffusion of molecules in the cytoplasm of the cells has long been of interest in the area of targeted drug delivery as well as for describing basic cellular processes. Diffusion of molecules through the cytoplasm or especially the nucleoplasm involves movement of relatively small, typically charged molecules through a sea of much larger, charged, molecules and supramolecular assemblies and is mainly affected by molecular crowding and charge-mediated binding. For small molecules such as metabolites and nucleic acids, an important parameter that describes the cytoplasmic rhelogy is the translational diffusion coefficient. In this work, we have developed a model system in which both molecular crowding and charge-mediated binding were addressed independently in a controlled manner. In particular, we obtained the translational diffusion coefficient of the positively charged protein, RNase A, in polymeric solutions of dextrans of various charges (which affects binding) and differing dextran concentrations (which affects crowding), as well as combinations of both. Using Fluorescence Correlation Spectroscopy (FCS), we observed that the diffusion of RNase A was unaffected by the presence of the positively charged or the neutral dextrans up to 20 μM dextran, above which concentration the diffusion was hindered by crowding. On the other hand, the presence of negatively charged dextrans slowed the protein diffusion significantly even at 0.2 μM dextran. The compound translational diffusion of RNase A decreased with increase in negative dextran concentration. The % bound RNase A also increased until it reached equilibrium binding of ∼90% bound RNase A at 14 μM dextran. Binding of RNase to the negatively charged dextrans was further confirmed by ultrafiltration. In addition, exact equilibrium dissociation constants were determined.