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In many fields, the goal is to obtain structures with small dimensions in the order of micro/nanometers. Small-sized systems can have countless applications in various industries such as cosmetology, medicine, and nutrition technology. Many techniques are used to obtain the most miniature possible spheres, such as interference with the composition, use of surfactants, or mechanical interference: rapid mixing, increased pressure, ultrasound. The use of ultrasound in the development of colloidal systems can be an effective method of reducing size of particles of dispersed phase and influencing the functions they represent. An important aspect here is the time during which the ultrasound is used. In this work, the influence of ultrasound on the chemosensory properties and size of produced ion-sensitive microspheres was investigated and compared. The chemosensory response of the developed microspheres was studied using spectrophotometry and spectrofluorimetry, while the size of the microsphere optodes was estimated by confocal microscopy.