•A broth dilution procedure for AST of Brachyspira species is described.•The broth dilution method was validated in a ring trial.•Intra- and inter-laboratory reproducibility was good.•New control strains are proposed.•Increased MIC corresponded to genomic data indicating decreased susceptibility. Brachyspira hyodysenteriae and Brachyspira pilosicoli cause economically important enteric disease in pigs. Treatment of these infections often includes antimicrobial administration, which can be most effective when therapeutic options are informed by antimicrobial susceptibility testing data. Here we describe a method for broth dilution antimicrobial susceptibility testing of these bacteria, both of which are difficult to culture in vitro. The protocol was evaluated for its fitness for use in an inter-laboratory ring trial involving eight laboratories from seven countries, and employing eleven test strains (5 Brachyspira hyodysenteriae including the type strain B78T and 6 Brachyspira pilosicoli) and six antibiotics. Overall intra- and inter-laboratory reproducibility of this method was very good (>90 % MICs at mode +/- 1 log2). Whole genome sequencing revealed good correspondence between reduced susceptibility and the presence of previously defined antimicrobial resistance determinants. Interestingly, lnu(C) was identified in B. pilosicoli isolates with elevated MICs of lincomycin, whilst tva(B) was associated with elevated MICs of pleuromutilins in this species. We designated two new control strains with MICs lying within currently tested ranges, including for the pleuromutilins, in contrast to the control strain B. hyodysenteriae B78T. These were deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The validation of a standard protocol and identification of new control strains facilitates comparisons between studies, establishment of robust interpretative criteria, and ultimately contributes to rational antimicrobial use when treating infected livestock.