Rejuvenators are used to restore the properties of reclaimed asphalt pavement (RAP) binders through readjusting the balance between the asphaltenes and maltenes fractions. To properly understand the effect of rejuvenators, it is important to analyze the chemical changes in the rejuvenated binders and to relate these changes to the rheological properties of the binders. In this study, RAP binders were blended with a bio-based and an aromatic extract petroleum-based rejuvenator additive. Chemical and thermal techniques such as differential scanning calorimetry (DSC), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and saturates, aromatics, resins, and Asphaltene Determinator (SAR-ADTM) were used to analyze the control and rejuvenated RAP binders. Glass transition temperatures were obtained using DSC and correlated with rheological measurements using a dynamic shear rheometer (DSR) and a 4-mm plate geometry. From FTIR results, the evolution of the carbonyl and sulfoxide indices with aging were determined and correlated with the complex shear modulus DSR measurements at different aging stages. The SAR-AD analysis was used to determine the changes in the asphalt fractions with the addition of the rejuvenators. Temperature-frequency DSR sweeps and bending beam rheometer (BBR) testing were conducted, and master curves were constructed for the different binders. From these master curves, the effect of the different rejuvenators on the cross-over temperature, glass-transition temperature, and the intermediate region temperature range (TIR) was determined.