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An ultra high-performance liquid chromatographic (UHPLC) method with PDA detection was developed and validated for the simultaneous quantification of meropenem, linezolid, and levofloxacin in human plasma and applied in human plasma of critical care patients. A semi-automated microextraction by packed sorbent (MEPS) for sample preparation was used. All parameters in the extraction step (pH, sample volume, sample dilution and number of aspiration – ejection cycles) and in the desorption step (percentage of acetonitrile in the solvent of elution and number of aspirations of elution solvent through the device) were statistically significant when the recovery was used as response. The method showed good linearity with correlation coefficients, r2 > 0.9991 for the three drugs, as well as high precision (RSD%< 10.83% in each case). Accuracy ranged from −7.8% to +6.7%. The limit of quantification of the three drugs was established at 0.01 μg/mL for linezolid and levofloxacin and 0.02 μg/mL for meropenem. Linezolid, meropenem, levofloxacin and the internal standard were extracted from human plasma with a mean recovery ranged from 92.4% to 97.4%. During validation, the concentration of meropenem, linezolid and levofloxacin was found to be stable after 3 freeze-thaw cycles and for at least 24 h after extraction. This method will be subsequently used to quantify the drugs in patients to establish if the dosage regimen given is sufficient to eradicate the infection at the target site.A novel sampling and clean-up procedure using microextraction by packed sorbent.UHPLC-PDA method development and validation.First MEPS-UHPLC-PDA method for determination of meropenem, levofloxacin, linezolid in human plasma.Therapeutic drug monitoring in critical care patients.