Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting around 35 million people worldwide. Cerebrospinal fluid (CSF) biomarkers entered the diagnostic criteria as support for early diagnosis. The classical biochemical signature of AD includes total tau (T-tau), phosphorylated tau (P-tau), and the 42 amino acid peptide (Aβ₄₂) of amyloid-β. Recent observations suggest that the use of CSF Aβ₄₂:Aβ₄₀ ratio rather than CSF Aβ₄₂ alone could contribute to reduce inter-laboratory variation in Aβ values and increasing diagnostic performance of the CSF AD biomarkers in routine practice. However, research efforts aimed at enriching the CSF biomarker panel are ongoing. The CSF AD signature is also crucial for the design of clinical trials for AD, since it best guarantees AD pathology as the cause of cognitive impairment. Accordingly, CSF biomarkers have been now reported in the inclusion criteria of Phase I, Phase II, and Phase III clinical trials as enrichment strategy. So far, one of the most important reasons for the failure of AD clinical trials was the inclusion of participants with unlikely AD pathology. In order to implement the use of CSF biomarkers in AD routine diagnostic work-up and as accepted strategy for enriching trial populations, inter-laboratory variability should be minimized. Increasing efforts should also be devoted to promote data sharing practices, encouraging individual participant data meta-analyses.