A specific and automated method was developed to quantitate the aminoglycosides amikacin, gentamicin, kanamycin, neomycin, paromomycin, and tobramycin simultaneously in human serum. Samples were prepared with an automated solid phase extraction (SPE). The hydrophilic interaction chromatography (HILIC) was used for separation of analytes from endogenous compounds and baseline separation. The aminoglycosides were detected with electrospray ionisation tandem mass spectrometry (ESI-MS-MS). Using a volume of 500 μl biological sample the lower limits of quantification were 100 ng/ml or better. The described HILIC-MS-MS method is suitable for therapeutic drug monitoring and for clinical and pharmcokinetical investigations of the aminoglycosides.