Raman spectroscopy has been used extensively to characterize the influence of mechanical deformation on microstructure changes in biomaterials. While traditional piezo-spectroscopy has been successful in assessing internal stresses of hard biomaterials by tracking prominent peak shifts, peak shifts due to applied loads are near or below the resolution limit of the spectrometer for soft biomaterials with moduli in the kilo- to mega-Pascal range. In this Review, in addition to peak shifts, other spectral features (e.g., polarized intensity and intensity ratio) that provide quantitative assessments of microstructural orientation and secondary structure in soft biomaterials and their strain dependence are discussed. We provide specific examples for each method and classify sensitive Raman characteristic bands common across natural (e.g., soft tissue) and synthetic (e.g., polymeric scaffolds) soft biomaterials upon mechanical deformation. This Review can provide guidance for researchers aiming to analyze micromechanics of soft tissues and engineered tissue constructs by Raman spectroscopy.