Proton transfer (PT) is a ubiquitous fundamental biological reaction. Directional PT reactions are commonly mediated by proteins in numerous biological processes, such as in photosynthesis and respiration . Since PT is essentially an acid-base equilibrium, the PT directionality, i.e., the proton motive force (PMF), is commonly modulated by a change in the protein structure during the PT process. To date, the main experimental approach to probe directional PT across biomolecules is to follow the end result of the PT process, which is usually the translocation of protons across a proton channel/pump or an enzymatic process. In some cases, the biomolecular PT process can be initiated with an external cue, such as with a light flash for light-gated proton channels or by depolarizing the membrane for voltage-gated proton channels, which enables following the kinetics of the PT process from start to end . The use of point mutations, chemical modifications, and inhibitors, together with the knowledge of the protein structure, has resulted in the understanding of PT pathways within proteins . In mechanistic terms, it is established that the PT pathway is comprised of specific amino acids that can participate in the formation of hydrogen bonds together with networks of water molecules within the protein . Nevertheless, the existing experimental approach to studying PT in biomolecules limits our ability to extract a generalized picture of the PT process from a desired point A to point B and to measure the associated timescales, which are commonly obtained from computational approaches . Here, we overcome this limitation by introducing a versatile donor-bridgeacceptor (D-B-A) system to induce and investigate PT pathways across peptides and proteins in space and time. We reveal the parameters needed for PT across peptides while deciphering the role of the side chain, the structure, and the environment in this process.