Hollow microspheres should be characterized in terms of physicochemical aspects to understand the flotation effect principle. There have been insufficient studies on the effect of hollow microspheres in water treatment in terms of flotation. In this study, various analytical and experimental approaches were utilized to identify the flotation characteristics and understand the effect of hollow microspheres on flotation. These approaches included analytical methods such as particle count and zeta potential measurements, scanning electron microscopy-energy dispersive X-ray spectroscopy analysis, X-ray photoelectron spectroscopy, a floc breakage experiment, and a collision efficiency model. The hollow microspheres were spherical shape with an average size of 50 μm. The microspheres were identified to have a higher negative charge (−69.1 mV) than microbubbles (−51.1 and −30.5 mV). The binding energy of Si–O from the hollow microspheres showed the highest peak at 103.18 eV and 61,503.7 counts/s. Si–O binding structure and the molecular structure of the SixOx series were considered to be a structure in which Si32− is bonded to oxygen ion. The optimum floc breakage condition was determined to be 15 min. The number of particles increased because the average particle size is increased with the concentration of hollow microspheres injection. The turbulent flocculation (TF) model was confirmed to be consentient to the experimental data in comparison with the white-water bubble blanket (WWBB) model. As a result, the hollow microspheres had the characteristic of increasing floc size so that flocs can be floated easily.