A transmembrane difference in the electrochemical potentials of protons (ΔμH+) serves as a free energy intermediate in energy-transducing organelles of the living cell. The contributions of two components of the ΔμH+ (electrical, Δψ, and concentrational, ΔpH) to the overall ΔμH+ value depend on the nature and lipid composition of the energy-coupling membrane. In this review, we briefly consider several of the most common instrumental (electrometric and EPR) methods for numerical estimations of Δψ and ΔpH. In particular, the kinetics of the flash-induced electrometrical measurements of Δψ in bacterial chromatophores, isolated bacterial reaction centers, and Photosystems I and II of the oxygenic photosynthesis, as well as the use of pH-sensitive molecular indicators and kinetic data regarding pH-dependent electron transport in chloroplasts, have been reviewed. Further perspectives on the application of these methods to solve some fundamental and practical problems of membrane bioenergetics are discussed.