Detection of carbapenemase-producing bacteria directly from blood cultures is a major challenge, as patients with bacteraemia are critically ill. Early detection can be helpful for selection of the most appropriate antibiotic therapy as well as adequate control of outbreaks. In the current study, a novel matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF)-based method was developed for the rapid, automated detection of carbapenemase-producing Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii directly from blood cultures. Carbapenemase activity was determined in 30 min by measuring hydrolysis of imipenem (0.31 mg/mL) in blood cultures spiked with a series of 119 previously characterised isolates, 81 of which carried a carbapenemase enzyme (10 bla_KPC, 10 bla_VIM, 10 bla_NDM, 10 bla_IMP, 26 bla_OXA-48-type, 9 bla_OXA-23, 1 bla_OXA-237, 3 bla_OXA-24 and 2 bla_OXA-58)_. Twenty blood cultures obtained from bacteraemic patients carrying bla_OXA-48-producing isolates were also analysed using the same protocol. Analysis was performed using MALDI-TOF Biotyper^® Compass software, which automatically provides a result of sensitivity or resistance, calculated as the logRQ or ratio of hydrolysis of the antibiotic. This assay is simple to perform, inexpensive, time saving, universal for Gram-negative bacilli, and highly reliable (overall sensitivity and specificity of 98% and 100%, respectively). Moreover, the protocol could be established as a standardised method in clinical laboratories as it does not require specialised training in mass spectrometry.