A novel method for the determination of aminoglycosides by surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) with the aid of silver-coated gold nanoparticles (Au@AgNPs) has been developed. The Au@AgNPs with surface capped by anionic citrate were used as concentrating probes as well as matrices in SALDI MS. Adsorption of aminoglycosides onto the nanoparticles was mainly through electrostatic attraction. The aminoglycoside-adsorbed nanoparticles were directly characterized by SALDI MS after a simple washing. Using Au@AgNPs to preconcentrate the aminoglycosides from 500 microL buffer solution, the limits of detection (LODs) at signal-to-noise ratio of 3 were 3, 25, 15, 30, and 38 nM for paromomycin, kanamycin A, neomycin, gentamicin, and apramycin, respectively. This method was successfully applied to the determination of aminoglycosides in human plasma samples. The LODs of aminoglycosides in plasma samples were 9, 130, 81, and 180 nM for paromomycin, kanamycin A, neomycin, and gentamicin, respectively. Recoveries of aminoglycosides in plasma samples were about 80%.