We describe the development of a capillary electrophoresis method for the determination of gentamicin C-1, C-1a, C-2a, and C-2 components in human serum. Using a weak cation-exchanger with 20 mm phosphate buffer, pH 7.4, 200 mm borate buffer, pH 9.0, and ammonia/methanol, solid-phase extraction (SPE) of gentamicin components from the human sera was performed. The extract was derivatized with 1,2-phthalic dicarboxaldehyde/mercaptoacetic acid reagent. The derivatives were separated with a background electrolyte comprising 60 mm 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer at pH 9.5 containing 31.6% m/v methanol, and quantified with UV-light absorption detection at 230 nm. The identity of the gentamicin components was confirmed by mass spectrometry. The SPE recovery of the gentamicin ranged from 78% to 93%. The calibration curves were linear from the concentration limit of quantitation (LOQ) to 30 mg/L for the gentamicin mixture. The LOO for gentamicin C, was 0.33 mg/L, for C-2a 0.23 mg/L, C-2 0.25 mg/L, C-1a 0.27 mg/L and the concentration limit of detection (LOD) for C-1 was 0.15 mg/L, C-2a 0-11 mg/L, C-2 0.12 mg/L, C-1a 0.13 mg/L. Intra-assay relative standard deviation (RSD) values were for C-1 (5%), C-1a (7%), C-2 (6.5%) and C-2a (9%); inter-assay RSD values were for C-1 (11%), C-1a (13.3%), C-2 (15%) and C-2a (14%). The Pearson's correlation between capillary electrophoresis and immunoassay revealed a linear relationship between these two techniques with r = 0.9. This method for determination of gentamicin C-1, C-1a, C-2a, and C-2 in human serum can thus be used in the entire therapeutic concentrations range of gentamicin.