SummaryWe provide a protocol using non-homologous end joining to integrate an oligonucleotide sequence of a fluorescence protein at theCDH1locus encoding for the epithelial glycoprotein E-cadherin. We describe steps for implementing the CRISPR-Cas9-mediated knock-in procedure by transfecting a cancer cell line with a pool of plasmids. The EGFP-tagged cells are traced by fluorescence-activated cell sorting and validated on DNA and protein levels. The protocol is flexible and can be applied in principle to any protein expressed in a cell line.For complete details on the use and execution of this protocol, please refer to Cumin et al. (2022).1