Human epidermal growth factor receptor 2 (HER2) overexpression occurs in various types of cancers. Regarding the anti-HER2 targeted therapies showed superior treatment outcomes in several (pre)clinical studies, we used multimodality image to rapidly select novel HER2-targeting antibodies for further therapeutics development. The four anti-HER2 antibodies (H32 IgG, 75 IgG, 61 IgG, and trastuzumab) labeled with either In-111 or a DyLight680 fluorescent dye were applied to perform cellular uptake, endocytosis, optical/microSPECT/CT imaging and biodistribution studies. In vitro and in vivo relative effectiveness of these antibodies were also compared in an N87 gastric cancer xenograft model. The internalized radioactivity of [111In]61 IgG in N87 cells increased from 33% at 12 hr to 56% at 48 hr after incubation, while the majority of other antibodies stayed on the cell membranes. Among these antibodies, 61 IgG showed the highest accumulation in tumors with the tumor-to-muscle ratio (T/M) of 131 ± 61.4 and 19.13 ± 3.42 conducted by IVIS and microSPECT/CT, respectively. We demonstrated that multimodality imaging is a reliable approach for selecting potential antibodies and found that 61 IgG manifested significant tumor accumulation with elevated internalization rate thus could be a suitable candidate for further development of new HER2-targeted therapies.