The creation of fuels, chemicals, and materials from plants can aid in replacing products fabricated from non-renewable energy sources. Before using biomass in downstream applications, it must be characterized to assess chemical traits, such as cellulose, lignin, or lignin monomer content, or the sugars released following an acid or enzymatic hydrolysis. The measurement of these traits allows researchers to gage the recalcitrance of the plants and develop efficient deconstruction strategies to maximize yields. Standard methods for assessing biomass phenotypes often have experimental protocols that limit their use for screening sizeable numbers of plant species. Raman spectroscopy, a non-destructive, non-invasive vibrational spectroscopy technique, is capable of providing qualitative, structural information and quantitative measurements. Applications of Raman spectroscopy have aided in alleviating the constraints of standard methods by coupling spectral data with multivariate analysis to construct models capable of predicting analytes. Hydrolysis and fermentation products, such as glucose and ethanol, can be quantified off-, at-, or on-line. Raman imaging has enabled researchers to develop a visual understanding of reactions, such as different pretreatment strategies, in real-time, while also providing integral chemical information. Finally, this review provides an overview of what Raman spectroscopy is, and how it has been applied to the analysis of whole lignocellulosic biomass, its derivatives, and downstream process monitoring.