Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a powerful technique for studying the diffusion behavior of macromolecules in solution. The diffusion coefficient and hydrodynamic radius depend on the size and shape of macromolecules. This review highlights the usefulness of DLS in studying the homogeneity of proteins, nucleic acids, and their complexes, as well as protein–small molecule interactions. DLS is also used as a complementary method to analytical ultracentrifugation studies and as a screening tool to validate solution scattering models using determined hydrodynamic radii. DLS is based on the measurement of Brownian motion of macromolecules in solution, which is related to their size and diffusion coefficient. The technique involves analyzing the intensity fluctuations of scattered light over time to determine the diffusion coefficient. The correlation function, G2(τ), describes the motion of macromolecules and can be used to calculate the hydrodynamic radius (Rh) using the Stokes–Einstein equation. DLS is particularly useful for determining the hydrodynamic behavior of proteins, nucleic acids, and viruses due to its ability to provide information on both size and aggregation. DLS has been widely used in biomedical sciences to study the homogeneity of biomolecular preparations, including proteins, nucleic acids, and their complexes. The technique is sensitive to even trace amounts of aggregation, making it a valuable tool for ensuring the quality of biomolecular preparations. DLS can also be used to study protein–protein interactions and protein–RNA interactions, providing insights into the conformation and behavior of these complexes in solution. The review discusses the theoretical foundations of DLS, including the relationship between the diffusion coefficient and hydrodynamic radius, as well as the different data analysis methods used to interpret DLS results. These methods include cumulant analysis and non-monomodal distribution methods, which are used to determine the size distribution of macromolecules and their homogeneity. In addition to studying homogeneity, DLS is also used to determine the size and conformation of RNA molecules. RNA molecules are highly labile and can be degraded by trace amounts of ribonuclease impurities, making DLS a valuable tool for ensuring the quality of RNA preparations. DLS has been used to study the homogeneity of viral RNA molecules, including the 5' and 3' terminal regions of the West Nile virus genome. Overall, DLS is a powerful and versatile technique that has been widely used in biomedical sciences to study the homogeneity, size, and conformation of macromolecules and their complexes. The technique provides reliable and rapid estimates of the quality of preparations and is particularly useful for ensuring the purity and homogeneity of biomolecular preparations.Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a powerful technique for studying the diffusion behavior of macromolecules in solution. The diffusion coefficient and hydrodynamic radius depend on the size and shape of macromolecules. This review highlights the usefulness of DLS in studying the homogeneity of proteins, nucleic acids, and their complexes, as well as protein–small molecule interactions. DLS is also used as a complementary method to analytical ultracentrifugation studies and as a screening tool to validate solution scattering models using determined hydrodynamic radii. DLS is based on the measurement of Brownian motion of macromolecules in solution, which is related to their size and diffusion coefficient. The technique involves analyzing the intensity fluctuations of scattered light over time to determine the diffusion coefficient. The correlation function, G2(τ), describes the motion of macromolecules and can be used to calculate the hydrodynamic radius (Rh) using the Stokes–Einstein equation. DLS is particularly useful for determining the hydrodynamic behavior of proteins, nucleic acids, and viruses due to its ability to provide information on both size and aggregation. DLS has been widely used in biomedical sciences to study the homogeneity of biomolecular preparations, including proteins, nucleic acids, and their complexes. The technique is sensitive to even trace amounts of aggregation, making it a valuable tool for ensuring the quality of biomolecular preparations. DLS can also be used to study protein–protein interactions and protein–RNA interactions, providing insights into the conformation and behavior of these complexes in solution. The review discusses the theoretical foundations of DLS, including the relationship between the diffusion coefficient and hydrodynamic radius, as well as the different data analysis methods used to interpret DLS results. These methods include cumulant analysis and non-monomodal distribution methods, which are used to determine the size distribution of macromolecules and their homogeneity. In addition to studying homogeneity, DLS is also used to determine the size and conformation of RNA molecules. RNA molecules are highly labile and can be degraded by trace amounts of ribonuclease impurities, making DLS a valuable tool for ensuring the quality of RNA preparations. DLS has been used to study the homogeneity of viral RNA molecules, including the 5' and 3' terminal regions of the West Nile virus genome. Overall, DLS is a powerful and versatile technique that has been widely used in biomedical sciences to study the homogeneity, size, and conformation of macromolecules and their complexes. The technique provides reliable and rapid estimates of the quality of preparations and is particularly useful for ensuring the purity and homogeneity of biomolecular preparations.