Giant plasma membrane vesicles (GPMVs) incorporating connexin proteins, referred to as connectosomes, serve as promising tools for studying cell membrane properties and intercellular communication. This study aimed to evaluate the membrane capacitance of connectosomes derived from HeLa cells and establish a method for assessing the gate function of connexin hemichannels. We investigated the behavior of dielectrophoresis (DEP) manipulation of connectosomes and HeLa cells by using microwell array electrodes. The frequency dependence of DEP force for connectosomes and HeLa cells suggested a low membrane capacitance of the connectosomes compared to that of HeLa cells. Positive DEP (p-DEP) was used to trap the connectosomes in the microwell array, where a relatively strong electric field was formed. This approach facilitated monitoring of the fluorescence intensity of individual connectosomes immediately after the solutions were exchanged, enhancing our ability to assess the release dynamics of fluorescent molecules and the hemichannel's open/closed states. The results confirmed that connexin hemichannels were regulated by the exterior concentration of Ca