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Carbapenemase resistance in <i>Enterobacterales</i> is a global public health problem and rapid and effective methods for detecting these resistance mechanisms are needed urgently. Our aim was to evaluate the performance of a MALDI-TOF MS-based “<i>Klebsiella pneumoniae</i> carbapenemase” (KPC) detection protocol from patients’ positive blood cultures, short-term cultures, and colonies in healthcare settings. Bacterial identification and KPC detection were achieved after protein extraction with organic solvents and target spot loading with suitable organic matrices. The confirmation of KPC production was performed using susceptibility tests and <i>bla</i>_KPC amplification using PCR and sequencing. The KPC direct detection (KPC peak at approximately 28.681 Da) from patients’ positive blood cultures, short-term cultures, and colonies, once bacterial identification was achieved, showed an overall sensibility and specificity of 100% (CI95: [95%, 100%] and CI95: [99%, 100%], respectively). The concordance between hospital routine bacterial identification protocol and identification using this new methodology from the same extract used for KPC detection was ≥92%. This study represents the pioneering effort to directly detect KPC using MALDI-TOF MS technology, conducted on patient-derived samples obtained from hospitals for validation purposes, in a multi-resistance global context that requires concrete actions to preserve the available therapeutic options and reduce the spread of antibiotic resistance markers.