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Linezolid is an important therapeutic option for the treatment of infections caused by multiresistant Gram-positive bacteria such as vancomycin-resistant Enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). However, the clinical benefit of linezolid is threatened by the emergence of resistant strains of MRSA and VRE reported in North America and Europe. For effective antimicrobial treatment, it is extremely important to have exact knowledge of drug concentrations at the site of action.A simple HPLC method for the rapid and precise determination of linezolid in different biomatrices (e.g., plasma, soft tissue, bone, dialysis fluid and used microbiological broth) was developed and validated. Proteins were precipitated with acetonitrile and separation was performed on a reversed-phase C8 column with a mobile phase consisting of water/ acetonitrile (80:20, v/v). UV detection was performed at 251 nm.This method has a lower limit of quantification of 0.3 μg/mL and a linear calibration range of 0.5–40 mg/mL. The method showed excellent reproducibility, with an inter- and intra-day assay precision of −5% (% relative standard deviation), as well as excellent accuracy, with inter- and intra-day assay accuracy ranging from 100.6% to 103.2%. Stability up to 6 months in water and plasma was proven. Quantitative recovery was possible after up to three freeze thaw cycles.The method is useful in the acquisition of in vivo and in vitro data. It is simple, flexible, specific, precise and reproducible, as well as of adequate sensitivity for clinical use.