The chelator mercaptoacetyltriglycine (MAG3) forms a single stable chelate with technetium-99m (^99mTc) oxotechnetate. The bifunctional N-hydroxysuccinimidyl ester of mercaptoacetyltriglycine with S-acetyl protection of the sulfhydryl group may be used to conjugate MAG3 to primary amine functionalized biomolecules for the purpose of radiolabeling with ^99mTc for gamma detection or single photon emission computed tomography imaging (SPECT). We report here an improved MAG3 conjugation and ^99mTc radiolabeling method capable of generating high radiochemical yield and high specific radioactivity. Post-labeling purification will not be needed if the protocol is followed as presented. Apart from the preparation of reagents, the conjugation and purification requires about 4 h, while the labeling with ^99mTc requires about an additional 30 min.