The HemosIL AcuStar Antiphospholipid assay (Instrumentation Laboratory, Bedford, USA) is a fully automated assay using chemiluminescent technology for the detection of anticardiolipin and anti-β2GPI antibodies. This assay showed excellent agreement between results of different laboratories. The cut-off values to define positivity were calculated in 250 healthy blood bank donors but were associated with large confidence intervals (CI). The objective of this study was to more precisely determine the cut-off values of the HemosIL AcuStar Antiphospholipid assay by increasing the number of healthy blood bank donors through a multicenter study and by applying a normalization procedure of the distribution of each antibody. Five laboratories participated to this study allowing the inclusion of 626 samples. We used a Box-Cox power transformation method to normalize the distribution and calculate the 99th percentile and the corresponding 95%CI for each antibody. The revised cut-off values were overall lower than those initially calculated with more stringent CI and yielded a 4.2%-increase of sensitivity with a 2.7% decrease of specificity regarding thrombotic events or obstetrical complications. We provide refined cut-off values for the detection of anticardiolipin and anti-β2GPI antibodies with the HemosIL AcuStar Antiphospholipid assay that should be preferred for routine use.