Abstract Background The differential identification of carbapenemases relies mostly on molecular techniques. Current phenotypic methods require 18 hours of incubation. We propose a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based direct-on-target microdroplet growth assay (DOT-MGA) aiming to offer an easy and rapid phenotypic identification of AmpC, KPC, MBL and OXA production. Methods Seven well-characterized Enterobacteriaceae strains recommended by EUCAST for carbapenemase detection were analyzed. Synergy between meropenem and carbapenemase inhibitors (phenylboronic acid, aminophenylboronic acid, cloxacillin, dipicolinic acid, ethylenediaminetetraacetic acid, and avibactam) and temocillin resistance were determined using a testing panel developed on a 96-spot MALDI-TOF MS target (MBT Biotarget 96, Bruker Daltonics, Germany). Microdroplets (6 µl) containing bacterial suspension and antibiotic or antibiotic/inhibitor in cation-adjusted Mueller–Hinton broth were spotted on the target and incubated for 4 hours at 36°C in a humidity chamber to avoid evaporation. The medium was subsequently removed and MALDI-TOF MS of the cells adhered to the target’s surface was performed. The minimum inhibitory concentration (MIC) was considered to be the lowest concentration at which the MALDI Biotyper software yielded no organism identification. Synergy was defined by an eightfold or greater reduction of the meropenem MIC in the presence of an inhibitor. The absence of synergy between meropenem and inhibitors as well as high-level temocillin resistance was considered suggestive of OXA production. Results were processed and interpreted with a computer-based algorithm. Results After 4 hours, the method was able to correctly detect the foreknown resistance mechanisms of all tested strains (KPC, MBL, OXA, and AmpC), yielding results that agreed with those obtained by performing broth microdilution with 18 hours of incubation. Conclusion The DOT-MGA approach allowed easy identification and differentiation of carbapenemase production, delivering reliable results one day earlier than the usual phenotypic methods, thus displaying great potential for the clinical setting. Disclosures E. A. Idelevich, Bruker Daltonik: Co-inventor of a pending patent, Licensing agreement or royalty and Speaker honorarium. K. Sparbier, Bruker Daltonik: Employee, Salary. M. Kostrzewa, Bruker Daltonik: Employee, Salary. K. Becker, Bruker Daltonik: Co-inventor of a pending patent, Licensing agreement or royalty and Speaker honorarium.