Antibiotic therapy is a crucial aspect of the management of hospitalized patients, however, current standard dosing protocols have been shown to often attain inadequate plasmatic concentrations which may impair the clinical outcome and promote the selection of multidrug-resistant bacteria. The aim of this study is to establish and validate a robust and fast liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the simultaneous analysis of four commonly used antibiotics (Meropenem, Piperacillin, Linezolid and Teicoplanin) in human plasma according to the European Medicines Agency (EMA) guidelines. Samples preparation was performed using a commercially available extraction kit which needs a very small amount of sample (50 μl). Antibiotics were detected, following a 7 min gradient separation, in multiple reactions monitoring (MRM) mode using a Qtrap 5500 triple quadrupole instrument equipped with an electrospray source operating in positive ion mode. The method, covering the antibiotics’ clinically relevant concentration ranges, is also able to quantify, individually, the major teicoplanin components. The high reproducibility and the need of a small amount of sample, associated with the use of a commercial kit, together with a short chromatographic time, makes the method particularly suited for high-throughput routine analysis. Monitoring of plasma antibiotic levels, as part of the clinical routine, would result in a quick therapy adjustment leading to a higher probability of eradicating the infection as well as a potential reduction of multidrug-resistance prevalence. The method was successfully applied to monitor the antibiotic concentration of 49 patients under therapy. © 2019 Elsevier B.V.