Simian virus 40 (SV40) is a possible vehicle for targeted drug delivery systems because of its low immunogenicity, high infectivity, and high transfection efficiency. To use SV40 for biotechnology applications, more information is needed on its assembly process to efficiently incorporate foreign materials and to tune the mechanical properties of the structure. We use atomic force microscopy to determine the effect of double-stranded DNA packaging, buffer conditions, and incubation time on the morphology and strength of virus-like particles (VLPs) composed of SV40 VP1 pentamers. DNA-induced assembly results in a homogeneous population of native-like, ∼45 nm VLPs. In contrast, under high-ionic-strength conditions, the VP1 pentamers do not seem to interact consistently, resulting in a heterogeneous population of empty VLPs. The stiffness of both in-vitro-assembled empty and DNA-filled VLPs is comparable. Yet, the DNA increases the VLPs’ resistance to large deformation forces by acting as a scaffold, holding the VP1 pentamers together. Both disulfide bridges and Ca2+, important in-vitro-assembly factors, affect the mechanical stability of the VLPs: the reducing agent DTT makes the VLPs less resistant to mechanical stress and prone to damage, whereas Ca2+-chelating EDTA induces a marked softening of the VLP. These results show that negatively charged polymers such as DNA can be used to generate homogeneous particles, thereby optimizing VLPs as vessels for drug delivery. Moreover, the storage buffer should be chosen such that VP1 interpentamer interactions are preserved.