RIG-I and MDA5 are the major intracellular immune receptors that recognize viral RNA species, undergo a series of conformational transitions and activate the interferon-mediated antiviral response. However, full-length RLRs resist crystallographic efforts and a molecular description of their activation pathways remains hypothetical. Here we employ HDX-MS to probe the apo states of RIG-I and MDA5 and to dissect the molecular details with respect to RNA recognition, ATP hydrolysis and CARDs activation. Our data provides high-resolution orthogonal views of RLR activation in solutions and offers evidence for the molecular mechanics of RLR activation. We show that human RIG-I maintains an auto-inhibited resting state owing to intra-molecular HEL2i-CARD2 interaction while apo MDA5 lacks the intra-molecular interactions and is fully extended. Our work also reveals the specific allosteric effects mediated by sequential addition of hairpin RNA, ATP and its analogs in respect to RIG-I CARDs activation. In contrast, polyIC (long duplex RNA) binds and activate RIG-I in a different manner. A high-resolution molecular model that refines the coorperative oligomerization of neighboring MDA5 molecules on polyIC RNA is also presented. Lastly, our example demonstrates that HDX-MS could provide unique structural insights into the large and dynamic macromolecular systems.