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Background: Alzheimer's disease (AD) is a complex disease that shares clinical features with other dementias. It is important to establish a specific and reliable diagnosis. Nowadays, AD diagnosis is based on cerebrospinal fluid (CSF) biomarkers. However, the corresponding cut-offs differ amongst studies. This study aims to evaluate the CSF biomarkers in the AD differential diagnosis., Methods: Clinical relevant biomarkers (amyloid β42 (Aβ42), t-Tau, p-Tau, amyloid β40 (Aβ40), neurofilament light chain (NfL)) were determined in CSF samples from participants classified as AD (n = 124) and non-AD (n = 148) patients from the Neurology Unit. They were included and evaluated consecutively (August 2018-October 2020). The clinical utility of these biomarkers was evaluated by AUC-ROC curves and the corresponding cut-off points were defined., Results: The results showed satisfactory accuracy (AUC-ROC 0.91 for Aβ42, 0.890 for t-Tau and 0.933 for p-Tau); whilst Aβ40 and NfL did not show good discriminatory capacity (AUC-ROC 0.557 and 0.738, respectively). The ratios Aβ42/Aβ40 and t-Tau/Aβ42 improved the diagnosis indices of each individual biomarker, with AUC-ROC of 0.980 and 0.971, respectively. Also, elevated levels of NfL were found in the frontotemporal dementia group compared with the other participant groups., Conclusions: The ratio Aβ42/Aβ40 showed the highest discriminating capacity between AD and non-AD patients and might be useful in clinical practice. Regarding NfL, it is not a specific biomarker for AD; however, it might be helpful for the differential diagnosis of frontotemporal dementia. Nevertheless, further analysis in an external cohort is required in order to validate these results., (© 2022 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)