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Introduction: Carbapenemase-producing Enterobacteriaceae (CPE) is a major global health threat, and development of rapid detection methods is desired. Here, we established a cost-effective and relatively rapid CPE detection method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)., Methods: We examined 134 CPE strains (IMP-type, NDM-type, VIM-type, KPC-type, OXA-48-like-type, and GES-type) and 107 non-CPE strains, previously confirmed by genetic tests. The proposed MALDI-TOF MS method involves mixing of a carbapenem drug [here, the commercially available imipenem (IPM) KB disc] and the bacterial strains to be tested, and the consequent drug hydrolysis owing to bacterial carbapenemase activity is confirmed by a waveform spectrum before and after 2 h of the mixing. As metallo-beta-lactamases require zinc in their active site, the false-negatives obtained from our method were cultured in presence of zinc sulfate solution and tested again., Results: Based on the presence or absence of the IPM (+cyano-4-hydroxy-cinnamic acid)-specific waveform peak near 489.45 m/z (±500 ppm), the detection sensitivity and specificity of our method for CPE were determined to be 94.8% and 91.6%, respectively. Seven false-negatives of IMP-type (4), VIM-type (2), and GES-type (1) were found, of which the IMP- and VIM-types tested positive as CPE after culture with zinc sulfate solution. Thus, the overall detection sensitivity improved to 99.3%., Conclusion: Our study proposes a new approach for CPE detection using MALDI-TOF MS. Moreover, we propose cultivation of test strains with zinc sulfate solution for efficient detection of IMP-type CPE, not only for MALDI-TOF MS, but also for other detection methods., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)