Effective rejuvenators that restore the thermo-mechanical properties of aged asphalt would extend pavement service life and increase utilization of recycled material, cutting material and environmental costs. However, the efficacy and mechanism of action need to be characterized in order to optimize rejuvenator formulation and dosage. This study introduces the RIPS method (Rejuvenation Index based on Polydispersity and Stiffness) which applies rheological characterization to build a simple model for examining and predicting the effect of rejuvenation on the rheology, polydispersity, and microstructure of aged asphalt binder. RIPS showed that addition of a specific bio-rejuvenator significantly improved the low-temperature properties of highly aged binder. The method accurately predicted the binder relaxation modulus and could calculate the exact rejuvenator dosage needed to restore the performance grade to specification. The rejuvenation mechanism was further examined using DSC, FTIR, and quantum level computational modeling, which found that polar chemical groups in the bio-rejuvenator could be responsible for solubilizing similar polar groups in aged binder leading to reduced molecular aggregate size and restoration of the thermo-mechanical properties of aged binder. These results provide insight into the molecular mechanisms of rejuvenation and offer a path towards more rational design and deployment of rejuvenator formulations.