Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, with the most common adult-onset neurodegenerative disorder affecting motoneurons. Although disruptions in macromolecular conformation and homeostasis have been described in association with ALS, the underlying pathological mechanisms are still not completely understood, and unambiguous biomarkers are lacking. Fourier Transform Infrared Spectroscopy (FTIR) of cerebrospinal fluid (CSF) is appealing to extensive interest due to its potential to resolve biomolecular conformation and content, as this approach offers a non-invasive, label-free identification of specific biologically relevant molecules in a few microliters of CSF sample. Here, we analyzed the CSF of 33 ALS patients compared to 32 matched controls using FTIR spectroscopy and multivariate analysis and demonstrated major differences in the molecular contents. A significant change in the conformation and concentration of RNA is demonstrated. Moreover, significantly increased glutamate and carbohydrates are found in ALS. Moreover, key markers of lipid metabolism are strongly altered; specifically, we find a decrease in unsaturated lipids and an increase in peroxidation of lipids in ALS, whereas the total amount of lipids compared to proteins is reduced. Our study demonstrates that FTIR characterization of CSF could represent a powerful tool for ALS diagnosis and reveals central features of ALS pathophysiology.