Non-pathogenic natural and recombinant strains of human Enteroviruses are the subject of ongoing study, with some strains having been approved for use as anticancer agents. The efficacy of on-colytic virotherapy is dependent upon the identification of the receptor utilized by a specific strain for cell entry and the presence of this receptor on the surface of cancer cells. Accordingly, a rapid and straightforward approach for determining the enteroviral receptors is necessary for the de-velopment of effective patient-specific virus-based cancer therapy. To this end, we created a panel of seven lines with double knockouts on the background of the HEK293T cell line, which lacks the IFNAR1 gene. In these lines, the main viral receptor genes, including PVR, CXADR, CD55, ITGA2, SCARB2, ICAM1, and FCGRT, were knocked out using the CRISPR/Cas9 system. A panel of lines was validated on a set of 12 Enteroviruses, providing a basis for studying the molecular mechanisms of enterovirus entry into cells and developing new therapeutic strains.