Replacing petroleum-based bitumen with alternative bio-based binders in asphalt mixtures is a promising solution for green transition of road construction industry. The mechanical performance of bio-extended bituminous binders and asphalt mixtures is not yet fully understood, in particular with respect to dynamic mechanical behavior due to traffic and environmental exposures. As bio-binders differ in their chemical compositions, their physical properties and mechanical behavior may vary and differ significantly from bitumen-based materials. This paper aims to contribute to this important topic by evaluating a spherical indentation test as a quasi-nondestructive tool for viscoelastic characterization of the asphalt mixture containing bio-extended bituminous binder. Asphalt indentation tests are more sensitive to the binder phase properties as compared to standard macro-scale asphalt tests and may thus be a potentially useful tool for monitoring binder properties from the measurements performed on asphalt mixtures without extracting the binder.