A high-performance liquid chromatographic (HPLC) method with UV and DAD detection for the quantitative determination of linezolid in human serum was developed in present work. Chromatography was carried out by reversed-phase technique on a RP-18 column with a mobile phase composed of 50 mM phosphate buffer and acetonitrile (76 : 26, v/v), adjusted to pH 3.5 with orthophosphoric acid. Serum samples were deproteinized with methanol centrifuged and then, the supernatant was analyzed using HPLC procedure. No interference was observed at the retention times of linezolid from blank serum or ten commonly used antibiotics. A concentration range from 0.50 to 30.0 g/mL was utilized to construct calibration curves. The lower limit of detection was determined to be 0.1 μg/mL of serum for both detectors. The lower limit of quantification of 0.25 μg/mL (CV = 2.6%) was established for determination using HPLC-UV and 0.5 μg/mL (CV = 5.42%) for HPLC-DAD. The recovery of linezolid was approximately 100%. Intra-day accuracy ranged from 0.97 to 12.63% and 0.74 to 10.85% for HPLC-UV and HPLC-DAD method, respectively. Intra-day precision was less than 4.69% for HPLC-UV and less than 5.42% for HPLC-DAD method. Tests confirmed the stability of linezolid in serum during three freeze-thaw cycles and during long-term storage of frozen serum for up to 6 weeks; in extracts it was stable in the HPLC autosampler over 24 h. Statistical analysis by Student's t-test showed no significant difference between the results obtained by these two methods. In summary, these methods will be used and adapted for infected patients in intensive care unit, to determine linezolid serum concentrations in order to know the pharmacokinetic profiles of linezolid.