Secondary structures, such as α-helices or β-sheets, play a crucial role in maintaining protein stability and enhancing functional properties, resulting in the resource of particular interest to structure—function relationships analysis. Fourier transform infrared (FT-IR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides, which is a widely utilized tool for determining secondary structures by analyzing the amide I band. FT-IR spectroscopy exhibits exceptional versatility and adaptability when applied to powdered samples with high absorption, as it remains unaffected by artifacts resulting from the scattering of radiation by sample particles. In contrast, circular dichroism (CD) spectroscopy, a traditional technique, is employed for the characterization of protein secondary structures in solution. This chapter provides an overview of the fundamental procedures involved in obtaining and interpreting FT-IR and CD data, along with techniques for analyzing spectra to estimate the composition of protein secondary structures.