This study investigates the chemical characteristics of rejuvenators and the effect of different rejuvenators on the morphology and fundamental behavior of rejuvenated asphalt binders. Gas Chromatography-Mass Spectroscopy (GC-MS) and atomic force microscopy (AFM) were conducted to determine the chemical composition of rejuvenators and asphalt surfaces’ morphology. Also, surface free energy (SFE) measurements were performed to quantify the cohesive bond energy of rejuvenated binders. The thin film oven test (TFOT) aged PG 58-28 binder was rejuvenated with waste cooking oil (UT), chemically modified waste cooking oil (TR), and Hydrolene (HL) at concentrations of 3%, 6%, and 9% by weight of the total binder. To understand the compatibility, styrene-butadiene-styrene (SBS) was also blended with rejuvenated binders. The experimental study found the variability of free fatty acid compositions in rejuvenators which is hypothesized to affect pavement performance. Results showed that rejuvenation alters the surface microstructure of binders, which provides insights into the overall performance of the binder. Also, rejuvenation improves the moisture damage resistance of binders significantly. This experimental study found a good correlation between the chemical, morphological and fundamental behavior of the rejuvenated binders, which is expected to help quantify the performance of rejuvenated asphalt mixes.