One of the clinically significant molecular targets in oncotheranostics is the receptor tyrosine kinase HER2 (HER2/neu, ERBB2), which is involved in the activation of various cellular programs. The overexpression of this receptor leads to uncontrolled cell proliferation, the initiation of oncogenesis, and is considered one of the most important oncogenic biomarkers. In vitro studies utilizing various HER2-positive cancer cell lines play a crucial role in the development of anti-HER2 targeted drug formulations. These cell lines differ in their structural and metabolic features, as well as in their sensitivity to hormones and other factors; therefore, the selection of an optimal cellular control is essential for the successful testing of HER2-specific agents. In the present study, a deletion in the ERBB2 gene was generated using CRISPR/Cas9 technology, with the success of the editing confirmed by sequencing of the target locus. A reduction in ERBB2 mRNA levels was demonstrated in three cancer cell lines with varying baseline HER2 receptor levels, alongside an alteration in the receptor's functional activity on the cell surface. Assessment of the binding efficiency of a fluorescently labeled HER2-specific antibody to the generated cell clones revealed a decrease in fluorescence intensity by 80.6-fold, 33.7-fold, and 2-fold in the SK-BR-3, SK-OV-3, and A549 cell lines, respectively. The generated cell lines with ERBB2 deletion represent a key tool for testing targeted therapeutics and can be utilized in the development of treatment modalities aimed at HER2-overexpressing malignant neoplasms