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Objectives The purpose of this study was to assess the feasibility of dual-energy CT-based material decomposition using dual-X-ray spectra information to determine local concentrations of holmium microspheres in phantoms and in an animal model. Materials and methods A spectral calibration phantom with a solution containing 10 mg/mL holmium and various tube settings was scanned using a third-generation dual-energy CT scanner to depict an energy-dependent and material-dependent enhancement vectors. A serial dilution of holmium (microspheres) was quantified by spectral material decomposition and compared with known holmium concentrations. Subsequently, the feasibility of the spectral material decomposition was demonstrated in situ in three euthanized rabbits with injected (radioactive) holmium microspheres. Results The measured CT values of the holmium solutions scale linearly to all measured concentrations and tube settings (R2 = 1.00). Material decomposition based on CT acquisitions using the tube voltage combinations of 80/150 Sn kV or 100/150 Sn kV allow the most accurate quantifications for concentrations down to 0.125 mg/mL holmium. Conclusion Dual-energy CT facilitates image-based material decomposition to detect and quantify holmium microspheres in phantoms and rabbits. Key Points • Quantification of holmium concentrations based on dual-energy CT is obtained with good accuracy. • The optimal tube-voltage pairs for quantifying holmium were 80/150 Sn kV and 100/150 Sn kV using a third-generation dual-source CT system. • Quantification of accumulated holmium facilitates the assessment of local dosimetry for radiation therapies.