In lung cancer, Vimentin intermediate filament (IF) proteins have been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-to-mesenchymal transition (EMT/MET), and the metastatic spread of cancer. Vimentin expression is generally upregulated when the cell is in the mesenchymal relative to the epithelial status. Here, we created a Vim-RFP reporter cell line (CCL-185EMT) using the CRISPR/Cas9 gene editing platform and the parental A549 (CCL-185) non-small cell lung cancer (NSCLC) cell line (a gold standard for studying EMT in cancer metastasis within the lung cancer research community). The created CCL-185EMT cell line habors a C-terminal red fluorescent protein (RFP) tag on the vimentin gene. This will enable the tracking of the EMT status of cells in vitro by monitoring RFP expression. The integrity of the Vim RFP knock-in has been verified at the genomic, mRNA and protein level for sequence and expression. Functional evaluation of CCL-185EMT shows sensitivity to anti-EMT drugs PP1 and A83-1. This provides the foundation for the high throughput (HTS) identification of new anti-EMT drugs for metastatic NSCLC therapy. The A549 Vim RFP reporter cell line provides a visualizable, convenient and sensitive platform for research on the mechanisms of metastasis in vitro and the development of new antitumor drugs for metastatic NSCLC.