Our lab and others have recently demonstrated that loading chemotherapeutics into DNA origami nanostructures for delivery can circumvent drug resistance mechanisms. While these and other studies have demonstrated exciting potential of DNA origami structures for biomedical applications, studies exploring function of DNA origami structures in physiological conditions are limited. A critical step towards clinical translation is understanding the stability of DNA origami nanostructures in physiological conditions. For drug delivery applications in particular, it is essential to quantify the ability of DNA origami structure to incorporate drugs and then retain them in physiological conditions. To address these key steps, we performed stability analysis of various DNA origami nanostructures in physiological conditions and investigated their ability to retain the chemotherapeutic drug daunorubicin, which is widely used in the treatment of acute leukemias. DNA nanostructures with various design parameters such as surface area, lattice type, and the inclusion of overhangs (short segments of ssDNA protruding from the structure) were tested to observe structure degradation in environments of varying salt (MgCl2) and fetal bovine serum (FBS) concentrations. We quantified stability through gel electrophoresis and verified structural integrity through transmission electron microscopy (TEM). We further used a spectrophotometer to quantify loading of daunorubicin into the nanostructures at different ratios to determine loading efficiencies at physiological temperatures. Intercalation was also verified by observing global twisting of structures via TEM. All structures tested were stable down to ~3mM MgCl2 for 24 hours and exhibited partial degradation in the range of 0-1 mM MgCl2. Nanostructures with square lattices and smaller surface areas were more resistant to salt degradation than those with a honeycomb lattices and larger surface areas. After incubating in FBS for 24 hours, nanostructures with square lattice designs and smaller surface areas showed continual stability even at 100% FBS. Results from daunorubicin experiments with a 24-hour incubation period indicate the nanostructures with larger surface area and overhangs resulted in higher base pair binding ratios. The results suggest that DNA origami is capable of withstanding cell conditions and retaining daunorubicin to varying degrees based on a structure’s design. This adds to the growing body of knowledge to enable effective design of DNA origami for drug delivery applications. To further explore the potential of DNA origami for drug delivery, a long-term objective is to apply the information gathered from stability and drug retention experiments to rationally design an optimal drug delivery nanostructure to test in vivo.