The potential of gene therapy has not yet been realized, largely due to difficulties in the targeted delivery of DNA to tissues and cells. Lipid-based nanovectors are of potential use in gene therapy due to their ability to enhance fusion with cellular membranes and transport the large polyanionic DNA molecules into the cytoplasm. While the research to date has mainly focused on liposome-based vectors, recently, nonlamellar phases with more complex internal architectures based on hexagonal or cubic symmetry have received increasing research attention due to their fusogenic properties, which may promote uptake of the DNA into the cell. Herein, we have carried out a fundamental physicochemical study to systematically analyze the encapsulation and release of nonfunctional double-stranded (ds) DNA fragments within monoolein (MO)-based cationic lipid phases of cubic symmetry (cationic cubic phases) and their dispersed submicron particles (cationic cubosomes). MO-based cationic cubic phases, both as the bulk phase and cubosomes, were formulated using six different cationic lipids, and their nanostructure was characterized in a high-throughput manner by synchrotron small-angle X-ray scattering (SAXS). dsDNA encapsulation was confirmed using agarose gel electrophoresis, and the effect on the internal nanostructure, size, and morphology of the cubosomes was investigated using synchrotron SAXS, dynamic light scattering, and cryo-transmission electron microscopy. Synchrotron radiation circular dichroism confirmed that the structure of the dsDNA fragments was unaffected by encapsulation within the cationic cubosome. The use of commercially available dsDNA ladders consisting of a controlled mixture of dsDNA fragments allowed us to determine release rates as a function of fragment size in a reasonably high throughput manner. An improved understanding of the loading capacity and release profile of nonfunctional biomolecules in cationic cubosomes will assist in the design of novel lipid nanovectors for gene delivery.