Abstract Background Cerebrospinal fluid (CSF) biomarkers β-amyloid 1-42 (Aβ1-42 ), also expressed as Aβ1-42 :Aβ1-40 ratio, T-tau, and P-tau181P , have proven diagnostic accuracy for mild cognitive impairment and Alzheimer's disease (AD). How to use, interpret, and disclose biomarker results drives the need for standardization. Methods Previous Alzheimer's Biomarkers Standardization Initiative meetings discussed preanalytical issues affecting Aβ1-42 and tau in CSF. This second round of consensus meetings focused on issues related to clinical use of AD CSF biomarkers. Results Consensus was reached that lumbar puncture for AD CSF biomarker analysis be considered as a routine clinical test in patients with early-onset dementia, at the prodromal stage or with atypical AD. Moreover, consensus was reached on which biomarkers to use, how results should be interpreted, and potential confounding factors. Conclusions Changes in Aβ1-42 , T-tau, and P-tau181P allow diagnosis of AD in its prodromal stage. Conversely, having all three biomarkers in the normal range rules out AD. Intermediate conditions require further patient follow-up.