Current diagnostic methods for prion diseases in vivo only work in late stages of the disease when neurodegeneration is very advanced and irreversible. Therefore, the search for biomarkers that can detect the disease before the onset of clinical symptoms is necessary. For this purpose, high-throughput techniques are of great interest. Here we have used mass spectrometry for the study of the cerebrospinal fluid proteome in 30 animals: sheep affected with natural scrapie, both in preclinical and clinical stages, and healthy sheep as a control group. Interestingly, using DDA and SWATH proteomic assays, we found 46 proteins in the preclinical stage of the disease that were significantly altered (p<0.01) compared to healthy sheep, mainly associated with biological processes such as stress response and inflammatory response. Five of them were selected for validation by ELISA, having been found to be altered also in other human neurodegenerative diseases: synaptotagmin binding, cytoplasmic RNA interacting protein (SYNCRIP), involved in nucleic acid metabolism; phospholipase D3 (PLD3) and cathepsin D (CTSD), both related to lysosomal apoptosis; C4 complement, an element of the classical immune response; and osteopontin (SPP1), a proinflammatory cytokine. These proteins were found to be significantly increased in the preclinical stage of disease. All proteins maintained their levels in the clinical phase, except for CTSD, which concentration returned to basal levels in the clinical group. Further research is ongoing to explore their future potential as preclinical biomarkers of prion diseases.