RIG-I like receptors (RLRs) are a family of ATP dependent RNA helicases which detect viral RNA within the host cytoplasm. RIG-I (retinoic acid inducible gene-I receptor), in particular, senses dsRNA with a 5´-triphosphate overhang and, following a conformational rearrangement and release of its CARD domains, mediates the ultimate induction of type I interferons and pro-inflammatory cytokines. It has been shown in the activation assays that a 5´ppp-10mer dsRNA hairpin is able to activate the RIG-I, but a 5´ppp-8mer does not. There is no biophysical evidence yet, however, of CARD release by the 10mer dsRNA and not the 8mer. We therefore investigated this using size-exclusion chromatography–coupled small-angle X-ray scattering (SAXS), as well as limited tryptic digest experiments and by using different ATP analogues we examined the importance of ATP on the conformational changes of RIG-I:RNA complexes. From SAXS data of RIG-I:RNA complexes we report that upon binding with 10mer RIG-I becomes extended with greater flexibility reflecting the release of its CARDs. This effect was not as great upon 8mer binding. Among the analogues tested, the addition of ADP-AlFx further assisted in the complete CARD release of RIG-I complexed with 10mer dsRNA. Tryptic digest profiles were also in accordance with the SAXS data. Our data are consistent with the minimal requirement of a 10mer for RIG-I activation, and the presence of ADP-AlFx further assisting complete release of RIG-I CARDs.