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Therapeutic drug monitoring (TDM) is a useful tool to optimize antibiotic therapy. Increasing interest in alternative dosing strategies of beta-lactam antibiotics, e.g. continuous or prolonged infusion, require a feasible analytical method for quantification of these antimicrobial agents. However, pre-analytical issues including sample handling and stability are to be considered to provide valuable analytical results.For the simultaneous determination of piperacillin, meropenem, ceftazidime and flucloxacillin, a high performance liquid chromatography (HPLC) method including protein precipitation was established utilizing ertapenem as internal standard. Long-term stability of stock solutions and plasma samples were monitored. Furthermore, whole blood stability of the analytes in heparinized blood tubes was investigated comparing storage under ambient conditions and 2–8 °C.A calibration range of 5–200 μg/ml (piperacillin, ceftazidime, flucloxacillin) and 2–200 μg/ml (meropenem) was linear with r2 > 0.999, precision and inaccuracy were <9% and <11%, respectively. The successfully validated HPLC assay was applied to clinical samples and stability investigations. At −80 °C, plasma samples were stable for 9 months (piperacillin, meropenem) or 13 months (ceftazidime, flucloxacillin). Concentrations of the four beta-lactam antibiotics in whole blood tubes were found to remain within specifications for 8 h when stored at 2–8 °C but not at room temperature.The presented method is a rapid and simple option for routine TDM of piperacillin, meropenem, ceftazidime and flucloxacillin. Whereas long-term storage of beta-lactam samples at −80 °C is possible for at least 9 months, whole blood tubes are recommended to be kept refrigerated until analysis.The presented method is a rapid and simple option for routine TDM of piperacillin, meropenem, ceftazidime and flucloxacillin in human plasma without extensive sample preparation and using common equipment, which is of advantage regarding time and consumables.Long-term storage of beta-lactam plasma samples at −80 °C is possible for 9–13 months, depending on the analyte, thus calibrators and quality control samples can be prepared in advance and stored deep frozen. These findings are of great interest for implementing analytical assays of beta-lactams in laboratory routine, facilitating daily measurements.Unprocessed whole blood tubes (S-Monovette Lithium-Heparin Gel, Sarstedt) directly after sampling are recommended to be kept refrigerated until analysis, as at room temperature, the compounds meropenem and piperacillin showed considerable degradation after 4 h and 6 h.