The quantification of antibiotics, using mass spectrometry, for monitoring therapeutic drugs is a key benefit in infection management. After an easy work-up of plasma samples, analysis were performed using both two widely used acquisition modes: MRM for the triple quadrupole spectrometer and fullMS/ddMS2 for the HRMS to quantify twelve antibiotics. Comparison between the two acquisition modes were performed. Validation parameters and sample values were used as comparison criteria. The results indicated a good correlation between the two methods, with an advantage for HRMS concerning the matrix effect. Both methods were applied to routine therapeutic drug monitoring. Display omitted •MRM and HRMS acquisitions were compared for the quantification of 12 antibiotics.•Assays were applied to routine therapeutic drug monitoring on both analytical systems.•HRMS showed better sensitivity for amoxicillin and cefazoline than MRM.•HRMS was less affected by the matrix effect than MRM.