In this work, we used Mössbauer spectroscopy as a new approach for experimental quantification of the self-diffusion coefficient (DMössbauer) and hydrodynamic (HD) size of iron-containing nanoparticles (NPs) in complex crowded solutions, mimicking cell cytoplasm. As a probe, we used 9 nm cobalt ferrite NPs (CFNs) dispersed in solutions of bovine serum albumin (BSA) with a volume fraction (φBSA) of 0-0.2. Our results show that the broadening of Mössbauer spectra is highly sensitive to the diffusion of CFNs, while when φBSA = 0.2, the CFN-normalized diffusivity is reduced by 86% compared to that of a protein-free solution. CFN colloids were also studied by dynamic light scattering (DLS). Comparison of the experimental data shows that DLS significantly underestimates the diffusion coefficient of CFNs and, consequently, overestimates the HD size of CFNs at φBSA > 0, which cannot be attributed to the formation of the BSA monolayer on the surface of CFNs.