Human epidermal growth factor receptor 2 (HER2) has been identified as a molecular target for breast cancer therapy, such as Trastuzumab (Herceptin®). This has been shown to improve patient survival substantially. The current study is aiming to locally produce an anti-HER2 monoclonal antibody (named Trastuzumab) which has an equivalent biological properties in comparison with the original version, Herceptin®). In silico design and construction of recombinant vectors, as well as the establishment of transfected cell lines with high expression of Trastuzumab were performed. Based on the protein sequences obtained from the Drugbank, the DNA sequences encoding for the light chain (Tras-Lc) and heavy chain (Tras-Hc) of Trastuzumab were optimized and integrated into pNanogen-Hygro and pNanogen-Puro vectors, respectively. The Neon Transfection System was used to co-transfect the pNanogen-Tras-Lc-Hygro and pNanogen-Tras-Hc-Puro constructs into CHO cells. Different co-transfected single-cell-colonies selected on media supplemented with hygromycin and puromycin were used for ELISA and SDS-PAGE assays to identify the CHO cell lines which highly express Trastuzumab. Based on the present results, 30μg of both constructs were suitable for DNA co-transfection. After 07 days of culture, the highest amount of Trastuzumab (561 µg/ml) was obtained from the H06LD68 cell line.