Background. Alzheimer cerebrospinal fluid (CSF) biomarkers have recently been included in the criteria for Alzheimer's disease (AD) diagnosis. Unfortunately, their wider use in routine and interpretation require more standardization, particularly for the pre-analytical steps. In particular, Aβ 42 peptide measurement is strongly influenced by the type of collection tube and by repeated freeze/thaw cycles. Objective. The objectives of this study were to compare, in clinical setting the impact of collection tubes and the repetition of freeze/thaw cycles on Aβ42 and Aβ40 concentrations and consequently determine if the Aβ42/Aβ40 ratio could resolve the diagnosis difficulties related to these pre-analytical parameters. Methods. CSF from 35 patients was collected in different PP and stored at-80°C after sampling. For CSF collected in the reference tube, three successive freeze-thaw cycles were done. Aβ42 and Aβ40 concentrations were determined in each condition in order to calculate the Aβ42/Aβ40 ratio. Results. Our results showed that CSF Aβ42 and Aβ40 values were significantly different according to the type of collection tube and the number of freeze/thaw cycles. Although the calculation of the Aβ42/Aβ40 ratio eliminated the effect of PP tube-dependent variation, this was not the case for freeze-thaw cycle-associated variation. Conclusion. The use of Aβ42/Aβ40 ratio rather than Aβ42 alone could contribute towards pre-analytical standardization, thus allowing the general use of CSF AD biomarkers in routine practice.