Linezolid is a promising drug for treating severe Gram positive bacterial infections of the eye. This is the first eye dedicated and validated high-performance liquid chromatography technique to measure linezolid in vitreous humor. The separation was performed by reversed phase chromatography. The mobile phase consisted of 25 mmol/L ammonium acetate-acetonitrile (76:24, v/v) (pH 5) with an Uvdetection procedure. The quantification limit in vitreous was 0.1 mg/L and the method was validated over a concentration range relevant for clinical use (0.2-20 mg/L). The intra- and inter-assay coefficients of variation were both satisfactory (below 8%). Stored at various concentrations for 4 weeks at +4°C and -80°C, linezolid in vitreous humor did not reveal any appreciable degradation. This technique, which already allowed us to study ocular pharmacokinetic of this antibiotic in an experimental rabbit model, could be suitable for human studies too.