Significance Proton diffusion (PD) across biological membranes is a fundamental process in many biological systems, such as in aerobic respiration or in the photosynthesis process. Even though this diffusion process has been studied for many decades, it is still highly enigmatic. Here, we suggest an experimental technique to study the very fast PD events taking place upon the photoinduced release of a proton on the surface of the membrane. We show that the diffusion, as well as the interaction between protons on the surface and protons in bulk media can be influenced by the nature of the membrane’s lipids. Our results shed some light on this unique PD process and help to overcome previous experimental contradictions in the literature.