A prerequisite for the use of dendrimers as drug delivery vehicles is the detailed molecular understanding of the drug interaction. The purpose of this study was to characterize the self-assembly process between siRNA and generation 7 poly(amidoamine) dendrimers and the resulting dendriplexes in aqueous solution using structural and calorimetric methods combined with molecular dynamics simulations. Complexes with a length scale of 150 nm showed a decreasing size with increasing amine-to-phosphate ratio by dynamic light scattering. At the molecular level, individual dendrimers studied by small-angle X-ray scattering (SAXS) showed no change in size upon siRNA binding, suggesting a rigid sphere behavior. Isothermal titration calorimetry (ITC) demonstrated exothermic binding with a concentration-dependent collapse of complexes. Both the experimentally determined ΔH(bind) and size were in close accordance with molecular dynamics simulations. This study demonstrates the unique complementarity of SAXS, ITC, and modeling for the detailed description of the molecular interactions between dendrimers and siRNA during dendriplex formation.