Studying changes in molecular conformations of cerebrospinal fluid (CSF) in neurological diseases and identifying disease-related secondary structure changes are promising potential biomarkers for early diagnosis and contribute to a better understanding of the pathophysiology. This protocol demonstrates that Fourier transform infrared spectroscopy (FTIR) characterization of CSF is a powerful analytical tool. When combined with chemometric analysis, infrared spectroscopy becomes an essential method for revealing central features of molecular structural changes, especially of protein secondary structure conformations associated with neurodegenerative diseases. This chapter describes a protocol for collecting FTIR spectra from CSF samples, and it covers instrumental options, sample preparation, and recent advancements in spectral data acquisition and interpretation.