Large conductance channels, when activated transiently, alter the plasma membrane permeability permitting the passage of molecules with the size of until 1000 Da. These pore-forming proteins are found in the plasma membrane of mammals and invertebrate cells such as the P2X7 receptor, transient receptor potential vanilloid 1 (TRPV1), transient potential melastin receptor 8 (TRPM8), pannexin-1, and hemichannel proteins. Other proteins may be secreted for organisms creating nonselective pathways to large molecules and proteins. Some examples are α-hemolysin and gramicidin. In both cases, an efficient manner to evaluate the functionality is using fluorescent dyes. Thus, we discuss the uses of the fluorescent dyes as tools to study the functionality and some biophysical properties of the proteins described above, among others.