This protocol describes the isolation of native protein complexes by density gradient ultracentrifugation. The outcome of an affinity capture and native elution experiment is generally a mixture of (1) the complex(es) associated with the protein of interest under the specific conditions of capture, (2) fragments of the complex generated by degradation or disassembly during the purification procedure, and (3) the protease or reagent used to natively elute the sample. To separate these components and isolate a homogeneous complex, an additional step of purification is required. Rate-zonal density gradient ultracentrifugation is a reliable and powerful technique for separating particles based on their hydrodynamic volume. The density gradient is generated by mixing low- and high-density solutions of a suitable low-molecular-weight inert solute (e.g., sucrose or glycerol). The gradient is formed in a solvent that could be any of the solvents used for the affinity capture and native elution and should help to preserve the structure and activity of the assembly.