Bacterial outer membrane vesicles (OMVs) are spherical membrane constructs shed by gram-negative bacteria. OMVs produced by the Lyme disease pathogen Borrelia burgdorferi have been identified to contain such virulence factors as OspA, OspB, OspC, and genetic material. However, the function and possible pathogenicity of borrelial OMVs are still undetermined. Therefore, further research on borrelial OMVs is required, and for that a standard method for OMV purification is necessary. Here we describe a successful and reproducible purification of borrelial outer membrane vesicles using concentration, filtration, and ultracentrifugation steps.