It has long been appreciated that the intracellular environment is highly crowded and a variety of approaches have been used to attempt to mimic this environment for in vitro studies. Recent studies have demonstrated that non-specific interactions between proteins play an important role in a variety of processes including rates of folding, protein stability and protein mobility and non-specific proteinprotein interactions are an important consideration in any studies involving macromolecular crowding. Previous diffusion studies have focused on small (<10 kDa) proteins and there is limited data on diffusion of larger proteins. This study extends previous work by using fluorescence anisotropy to measure rotational diffusion of 8 different proteins in the presence of a variety of macromolecular crowding agents. These studies demonstrate that non-specific interactions with bulk proteins impact rotational diffusion as a function of size and that the chemical nature of the bulk protein is more significant than the size of the bulk protein. Our work also shows that viscosity has very little impact on the rotational diffusion of each protein. While there is still much to learn about the chemical nature of the intracellular environment, this work demonstrates that the nature of the protein of interest and the environment surrounding the protein can significantly impact its rotational mobility.