This research seeks to interpret the component analysis of an innovative bio-asphalt binder using guayule resin in addition to crumb rubber modifier (CRM) at high concentrations. Such asphalt modification aims to minimize the dependency on virgin asphalt binder and provide new solutions concerning sustainable, flexible pavement industry. Guayule resin is a promising bioresource for asphalt binder replacement. By now, it could be considered a no value byproduct extracted during the guayule natural rubber production. CRM is a recycled material derived from scrap tires. The provided interpretation could help in understanding the asphalt-rubber-guayule interaction mechanism. Fourier transform infrared spectroscopy (FTIR), supported by thermo-gravimetric analysis (TGA), was used to investigate the component analyses of guayule resin composition, asphalt guayule interaction, and asphalt rubber guayule interaction, compared to corresponding asphalt rubber interaction. Additionally, the rheological properties at elevated temperatures were provided to link the microscale properties with the final product performance. The study clarified the distinct carbon and hydrogen compositional elements of guayule resin. Asphalt and guayule resin have similarities in chemical composition and rheological behavior with temperature susceptibility. The asphalt guayule binder had physical interaction. However, when both interacted with rubber, a chemical interaction was attributed, with no difference in rubber dissolution tendency, in asphalt rubber guayule, compared to asphalt rubber. A bio-binder composed of 62.5% asphalt, 25% guayule and 12.5% CRM had the potential to provide rheological properties better than base asphalt. Such behavior was interpreted by a high release of rubber components.