In the interdisciplinary field of chemistry, physics, and biology, single-molecule techniques delineate the individual behavior of molecules, decoding the mysteries of biophysical processes at the most fundamental level. Specifically, we utilize optical tweezers to manipulate a single molecule and apply forces on it to measure the corresponding molecular extensions as well as force responses. The single molecule in extension is characterized by UV-Vis absorption, Raman, and/or fluorescence, yielding spectra that reflect the chemical environment of the molecule’s functional groups, providing molecular-level mechanistic insights.