Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a powerful technique for studying the diffusion behavior of macromolecules in solution. This review highlights the usefulness of DLS in characterizing the homogeneity of proteins, nucleic acids, and complexes, as well as in studying protein-small molecule interactions. DLS provides valuable information on the diffusion coefficient and hydrodynamic radii, which are crucial for understanding the size and shape of macromolecules. The technique is particularly useful for determining the hydrodynamic behavior of proteins, nucleic acids, and viruses due to its ability to provide insights into both size and aggregation. The review covers the theoretical background of DLS, including the principles of light scattering and the relationship between particle motion and measured fluctuations. It also discusses the data analysis methods used in DLS, such as monomodal and non-monomodal distribution methods, and their applications in studying the size distribution of hydrodynamic radii. The review further explores the practical applications of DLS in detecting aggregation in recombinant proteins, studying protein-protein interactions, evaluating the homogeneity of viral RNA molecules, and characterizing protein-RNA interactions. Overall, DLS is a versatile tool that offers rapid and reliable measurements, making it an essential technique in biomedical sciences.Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a powerful technique for studying the diffusion behavior of macromolecules in solution. This review highlights the usefulness of DLS in characterizing the homogeneity of proteins, nucleic acids, and complexes, as well as in studying protein-small molecule interactions. DLS provides valuable information on the diffusion coefficient and hydrodynamic radii, which are crucial for understanding the size and shape of macromolecules. The technique is particularly useful for determining the hydrodynamic behavior of proteins, nucleic acids, and viruses due to its ability to provide insights into both size and aggregation. The review covers the theoretical background of DLS, including the principles of light scattering and the relationship between particle motion and measured fluctuations. It also discusses the data analysis methods used in DLS, such as monomodal and non-monomodal distribution methods, and their applications in studying the size distribution of hydrodynamic radii. The review further explores the practical applications of DLS in detecting aggregation in recombinant proteins, studying protein-protein interactions, evaluating the homogeneity of viral RNA molecules, and characterizing protein-RNA interactions. Overall, DLS is a versatile tool that offers rapid and reliable measurements, making it an essential technique in biomedical sciences.