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Background: detailed information about circulating tumor cells (CTCs) as an indicator of therapy response and cancer metastasis is crucial not only for basic research but also for diagnostics and therapeutic approaches. Here, we showcase a newly developed IsoMAG IMS system with an optimized protocol for fully automated immunomagnetic enrichment of CTCs, also revealing rare CTC subpopulations. Methods: using different squamous cell carcinoma cell lines, we developed an isolation protocol exploiting highly efficient EpCAM-targeting magnetic beads for automated CTC enrichment by the IsoMAG IMS system. By FACS analysis, we analyzed white blood contamination usually preventing further downstream analysis of enriched cells. Results: 1 µm magnetic beads with tosyl-activated hydrophobic surface properties were found to be optimal for automated CTC enrichment. More than 86.5% and 95% of spiked cancer cells were recovered from both cell culture media or human blood employing our developed protocol. In addition, contamination with white blood cells was minimized to about 1200 cells starting from 7.5 mL blood. Finally, we showed that the system is applicable for HNSCC patient samples and characterized isolated CTCs by immunostaining using a panel of tumor markers. Conclusion: Herein, we demonstrate that the IsoMAG system allows the detection and isolation of CTCs from HNSCC patient blood for disease monitoring in a fully-automated process with a significant leukocyte count reduction. Future developments seek to integrate the IsoMAG IMS system into an automated microfluidic-based isolation workflow to further facilitate single CTC detection also in clinical routine.