Aminoglycosides are antibiotics used mainly in the treatment of infections caused by aerobic gram-negative bacteria. The chemical structures of these compounds consist of two or more aminosugars joined by a glycosidic linkage to an aminocyclitol component. These compounds are very hydrophilic due to the large number of amino and hydroxyl groups in their structures. This presents a challenge when developing an HPLC/MS/MS method because the use of traditional reversed-phase HPLC will not retain these compounds on the column. Here we report on the development of an accurate, precise and specific HPLC/MS/MS assay for the determination of amikacin, neomycin and gentamicin from rat and mouse plasma as well as various tissues.