Background <p>Cell line authentication and karyotype assessment are two critical quality control tests that should be performed when using cell lines for biologic research and are expected measurements for cell therapy development. The current paradigm requires two separate measurements to assess these attributes, therefore a single straightforward approach that could assess karyotype and authenticate cell lines is beneficial.</p> Results <p>We have developed a new optical genome mapping based approach named OGM-ID which can authenticate cell lines utilizing the same data that has previously been demonstrated as an alternative for traditional karyotyping. OGM-ID utilizes genome wide large (&gt; 500&#xa0;bp) insertions and deletions to uniquely identify cell lines. OGM-ID can be used to determine interspecies and intraspecies contamination. Benchmarking of OGM-ID was performed using three different family lineages, where replicates were clearly identified, and the relative genetic distance between individuals could be further monitored utilizing the zygosity of variants. Additionally, the donor of wild type and edited iPSCs was correctly determined even after multiple clonal selection events. Current limitations of OGM-ID require control around the version of Bionano Solve used and similar depths of coverage between samples.</p> Conclusions <p>OGM-ID is a whole genome technique that produces results comparable to other cell line authentication techniques with the added benefit of obtaining a cell line’s karyotype simultaneously. OGM-ID’s ability to distinguish large insertions or deletions, which are common in genome editing, also gives it a unique ability to distinguish between multiple clonal iPSC-derived allogeneic cell product candidates derived from the same donor.</p>