As an important characterization technique, experimentally determined zeta potential values can be used to effectively predict the surface charge of colloidal particles and then estimate the strength of mutual repulsion or attraction between particles. The dispersion mechanism of molecules or particles within colloidal systems can also be elucidated by measuring the zeta potential. The information obtained by measuring this value is crucial for the intentional control of a colloidal system’s electrostatic dispersion. Moreover, the isoelectric point of protein can also be obtained by determining its zeta potential. Obtaining zeta potential values of any particle provides a vital foundation for understanding and modifying its surface electrical properties. At present, the measurement of zeta potential is widely used in food, cosmetics, health care, environmental protection, marine chemistry, and other industries. This chapter provides a comprehensive guide to instrumental methods for zeta potential determination in protein samples.