Chemistry in general concerns a change in substance or structure on various timescales. The mechanistic study is needed to decipher the actual process going from reactants to products, or commonly referred to, across the reactive potential energy surface. To obtain this “bottom-up” knowledge that bridges microscopic structure to macroscopic function, the molecular conformational dynamics leading to chemical reactivity need to be resolved both spatially and temporally. A powerful physical chemistry approach is femtosecond stimulated Raman spectroscopy (FSRS), which is an emerging structural dynamics tool to study aqueous molecules in situ on their intrinsic timescales (e.g. a vibrational period). Recent FSRS results on the photoacidity of pyranine (HPTS, 8-hydroxypyrene-1,3,6-trisulfonic acid) in acetate water will be discussed, in the context of excited state proton transfer and multidimensional reaction coordinate. The nascent acetic acid peak shows a delayed onset of ~300 fs compared with the deprotonated HPTS signal. Several key low-frequency modes are revealed to play an important role in guiding the photoacid proton through the hydrogen-bonding chain with various lengths, involving the acetate and/or water molecules as proton acceptors. FSRS can also shed light on the structural transition involved in the photoswitching of various fluorescent proteins.