Asphalt binder modification is an unending research topic that rouses the interest of many researchers worldwide. Many studies investigated the effect of implementing nanotechnology into asphalt pavement. The use of nanomaterials to enhance the rheological properties of asphalt binders is proven to be remarkably successful. Nanomaterials have unique properties, and with a small amount of modification, an important enhancement of asphalt performance can be attained. One of the main challenges of this application is the optimal mixing conditions to assure a thorough dispersion of the nanoparticles in the asphalt binder. This study focuses on the effect of different laboratory mixing conditions on the rheological properties of asphalt binders. Iron nanoparticles were used with two different concentrations: 2% and 6% by weight of asphalt binder. Investigated mixing conditions include mixing speeds (3000 and 6000 rpm), mixing time (60 min and 120 min), high shear mixing geometries (mechanical blade, dispersion blade, and three rotor-stator rings (slotted, round, and fine screen), mixing volume (2 liters and one liter), and effect of sonication. Physical and rheological properties of control and nano modified asphalt binders were investigated to assess the mixing conditions using a wide range of test methods including penetration, softening point, rotational viscosity, and Dynamic Shear Rheometer (DSR) for high-temperature performance using maximum temperature Performance Grade (PG) and Multiple-Stress Creep Recovery (MSCR) tests. In addition, Scanning Electron Microscopy (SEM) and image processing were used to investigate the dispersion of nanoparticles within the asphalt binders at different mixing conditions to determine the optimal laboratory mixing conditions. The findings of this research study showed that mixing at high rotational speed (6000 rpm) for longer period (2 hours) demonstrated the best asphalt performance and smallest particle sizes. Additionally, utilizing the mechanical and the dispersio