Background In April 2020, the BIOMILK 300 LAC method for the quantification of lactose in lactose-free and low lactose dairy products was adopted as First Action Official Method of Analysis 2020.09 by the AOAC INTERNATIONAL. In order to test the reproducibility of the method, as the last step towards the Final Action status, a collaborative study was organized by BIOLAN Microbiosensores. Objective Fifteen collaborators within the European Union took part in this study, where nine different samples were sent as duplicate blind test portions for lactose determination. The data obtained were used to determine method repeatability and reproducibility and also to validate the new version of the biosensor. Method The test method is based on the direct enzymatic recognition-electrochemical detection of trace levels of lactose over a wide range of dairy samples by means of the BIOMILK 300 biosensor, in less than five minutes, and without intricate sample pre-treatments. Results All samples resulted in a repeatability relative standard deviation (RSDr) of < 8.1% and a reproducibility relative standard deviation (RSDR) of 14.0% maximum, meeting requirements from SMPR 2018.09. Conclusions On the basis of these results, the enzymatic amperometric biosensor method developed by BIOLAN Microbiosensores was adopted as Final Action Official MethodSM in July 2022.