Vancomycin-resistant enterococci (VREs) have become one of the most important noso-comial pathogens worldwide, associated with increased treatment costs, prolonged hospital stays and high mortality. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. The lateral flow immunoassay NG-Test VanB (NG Biotech) was evaluated for the rapid detection of VanB-producing vancomycin-resistant enterococci (VanB-VREs) using 104 well-char-acterized enterococcal isolates. The sensitivity and specificity were both 100% when bacterial cells were grown in the presence of vancomycin used as a VanB inducer. The NG-Test VanB is an effi-cient, rapid and easy to implement assay in clinical microbiology laboratories for the confirmation of VanB-VREs from colonies. Together with the NG-Test VanA, they could replace the already ex-isting tests available for the confirmation of acquired vancomycin resistance in enterococci, especially from selective media or from antibiograms, with 100% sensitivity and specificity. Rapid detection in less than 15 min will result in more efficient management of carriers and infected patients. In addition, these tests may be used for positive blood cultures, given a 3.5 h sub-culturing step on Chocolate agar PolyViteX in the presence of a 5-µg vancomycin disk, which is routinely performed in many clinical microbiology laboratories for every positive blood culture for subsequent MALDI-TOF identification of the growing bacteria.