Global changes in the conformations of proteins in the presence of macromolecular crowding agents have been well documented. Here, we have used solvation dynamics to monitor the changes in a specific domain of the multidomain protein human serum albumin (HSA) in the presence of various crowders. The solvation probe 6-bromoacetyl-2-dimethylaminonaphthalene was site-specifically attached to the cysteine-34 of domain I of HSA. Analyses of the time resolved Stokes shift of this probe in the presence of crowding agents revealed a significant retardation of the solvent coordinate, particularly in a crowder-dependent manner. We attribute the observed slowing primarily to the increased internal protein friction in the presence of these polymers, implying considerable stiffness of the protein matrix. We have discussed our findings with regard to recent reports on cellular interiors and have also made an attempt to assess the importance of the physiological concentration of macromolecules in protein dynamics and function.