A specific, and automated method was developed to quantitate neomycin in human serum. Samples were prepared with an automated solid phase extraction (SPE). The hydrophilic interaction chromatography (HILIC) was used for additional sample cleanup and baseline separation. The analyte neomycin was detected with electrospray ionisation tandem mass spectrometry (ESI-MS-MS). Using a volume of 500 μl biological sample the lower limit of quantification was 100 ng/ml. The described HILIC-MS-MS method is suitable for clinical and pharmcokinetical investigations of neomycin.