Abstract We present a magnetic bead assay for rapid identification and isolation of single intact circulating tumor cells (CTCs). Cell viability is essential for cell culture and downstream molecular analysis. Many CTC isolation technologies, including CellSearch, uses fixation and fluorescent imaging for CTC detection that the captured CTCs are no longer viable. This novel assay utilize magnetic sensing for CTC identification, and therefore preserve CTC integrity and viability. The assay is performed on a microelectronic chip which also generates spatially-patterned microscopic magnetic field to sort single CTCs. We evaluated the performance metrics of the assay with EGFP-expression breast cancer patient derived CTC cells mixed with peripheral blood mononuclear cells (PBMCs) in vitro. Cell suspensions were mixed with magnetic microbeads coated with anti-EpCAM antibodies and then isolated with a magnet. Different amount (20, 50, 100, 500) of CTCs from serial dilution were spiked into 5x10^6 human PBMCs labelled with Alexa Flour 488 anti-human CD45 antibodies. After the assay, no PBMCs are visible which shows high purity in recovered CTCs. Recovery rate, defined by the ratio of the mean of the recovered CTC count to the mean of the spiked CTC count, is in the range of 87% to 92% for the various CTC spiked-in amount. In each spiking experiment, triplicate measurement and controls are used. Total assay time is less than 80 minutes. Conclusion: A rapid assay for isolation and detection of CTCs from PBMCs is demonstrated which shows high recovery rate and viability. This novel assay has great potential for single intact rare cell isolation. Citation Format: Lili Huang, Le Shen, Min Yu, Paul Liu. A novel assay for rapid identification and isolation of single intact circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3866.