Rituximab when radiolabelled with 177Lu or 90Y has been investigated for the treatment of patients with Non-Hodgkin's Lymphoma. In this study, we optimized the preparation of antibody conjugates with chelating agent in the freeze-dried kit. It shortens procedures needed for the successful radiolabeling with lutetium-177 and yttrium-90 and assures reproducible labelling yields. Various molar ratios of Rituximab:DOTA (from 1:5 to 1:100) were used at the conjugation step and different purification method to remove unbound DOTA were investigated (size-exclusion chromatography, dialysis, ultrafiltration). The final monoclonal antibody concentration was quantified by Bradford method, and the number of DOTA molecules was determined by radiolabeling assay using 64Cu. The specific activity of 177Lu-DOTA-Rituximab and 90Y-DOTA-Rituximab were optimized using various amounts of radiometal. Quality control (SE-HPLC, ITLC) and stability study were performed. An average of 4.2 ± 0.8 p-SCN-Bz-DOTA molecules could be randomly conjugated to a single molecule of Rituximab. The ultrafiltration system was the most efficient for purification and resulted in the highest recovery efficiency (77.2%). At optimized conditions the 177Lu-DOTA-Rituximab and 90Y-DOTA-Rituximab were obtained with radiochemical purity >99% and specific activity ca. 600 MBq/mg. The radioimmunoconjugates were stable in human serum and 0.9% NaCl. After 72 h of incubation the radiochemical purity of 177Lu-DOTA-Rituximab decreased to 94% but it was still more than 88.1% for 90Y-DOTA-Rituximab. The radioimmunoconjugate showed stability after six months storage at 2 - 80C, as a lyophilized formulation. Our study shows that Rituximab-DOTA can be efficiently radiolabeled with 177Lu and 90Y via p-SCN-Bn-DOTA using a freeze-dried kit.