Mesoporous alumina spherical particles, synthesized via spray-drying with the self-assembly of EOnPOmEOn, have been utilized for the development of clinical-grade molybdenum-99/technetium-99m (99Mo/99mTc) generators. When evaluated as molybdenum (Mo) adsorbents, the mesoporous alumina spherical particles are useful for effective adsorption of molybdenum ions rather than commercially available particulate alumina. The effects of surfactant removal methods on the Mo adsorption property are also systematically investigated using the batch method. Batch adsorption studies reveal practical adsorption capacities ranging from 45.9 to 91.2 mg Mo g-1 in a Mo solution (1000 mg Mo L-1) at pH 3. The experimental results indicate the following trend in Mo adsorption capacity: solvent extraction > calcination (400°C and 800°C) > commercially available alumina (Medical Al2O3 used as is). To explore the feasibility of developing a clinical-scale generator, a novel tandem column generator concept is employed. Using the spray-dried and extracted mesoporous alumina, 99mTc eluted from the generator exhibits high radionuclidic, radiochemical, and chemical purity, making it suitable for the preparation of 99mTc-labeled radiopharmaceuticals.