A high-sensitivity benchtop x-ray fluorescence (XRF) imaging system, based on a high-power x-ray source and silicon drift detector, has been developed. This system allows gold L-shell XRF-based quantitative imaging of gold nanoparticles (GNPs) at concentrations as low as 0.007 mg/cm3 (7 ppm) in biological tissues/water. Its capability for biomedical applications was demonstrated by imaging the GNP distribution within a small (∼12×11×2 mm3) ex vivo sample (extracted from a murine tumor after intravenous GNP administration). The results suggest direct translatability for routine preclinical ex vivo imaging tasks involving GNPs, as well as the possibility for in vivo imaging of small/superficial animal tumors.