The biological functions of the cell membrane are regulated by its physicochemical properties, such as the transmembrane potential, phase state and hydration. Therefore, the in situ investigation of these properties is attracting a rising interest in the field of membrane biophysics. In these regards, a wide variety of fluorescent membrane probes has been developed, providing powerful tools for fluorescence microscopy and/or fluorescence spectroscopy to study the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order and electrostatic potential at the sites of their location. Despite the success of this approach, further developments rely on the design of fluorescence probes with optimized photophysical properties. This review discusses the most successful fluorescent molecular probes and their application to the study of the plasma membrane viscosity, lipid order and potential.