A high-performance liquid chromatographic method for the determination of linezolid in human plasma was developed and validated. After precipitation of plasma proteins with perchloric acid, the protein-free supernatant was separated by isocratic reverse-phase chromatography on a X Bridge C18 column. The mobile phase consisted of a mixture of phosphoric acid 0.05%: acetonitrile (75:25, v/v) with a flow rate of 1 mL/min. The column elute was monitored at 254 nm. The method was linear from 0.2 to 48 mg/L (mean r2 = 0.9996, n = 10). The observed intra- and inter-day assay imprecision ranged from 2.83% to 8.16% (18.80% at the lower limit of quantification); inaccuracy varied between -0.33% and 8.18%. Mean drug recovery was 99.8% for linezolid and 90.0% for the internal standard (para-toluic acid). The method was found to be precise and accurate and suitable for therapeutic drug monitoring of linezolid in routine clinical practice.