Lignin in plant cell wall is a complex amorphous polymer and is biosynthesized mainly from three aromatic alcohols, namely, p-coumaryl, coniferyl, and sinapyl alcohols. This biosynthesis process consists of mainly radical coupling reactions and creates a unique lignin polymer in each plant species. Generally, lignin mainly consists of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units and is linked by several types of carbon-carbon (β-β, β-5, β-1, and 5–5) and ether bonds. Due to the structural complexity, various molecular spectroscopic methods have been applied to unravel the aromatic units and different interunit linkages in lignin from different plant species. This chapter is focused on the application of ultraviolet (UV) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, Fourier transform Raman (FT-Raman) spectroscopy, fluorescence spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy to lignin structural elucidation.