Abstract Circulating tumor cells (CTCs), released into the bloodstream from primary and metastatic cancers, are valuable tools for understanding tumor biology. However, since they are rare, their detection is hampered by low efficiency and a lack of standardization in current technologies. To overcome these problems, we used Imaging Flow Cytometery along with fluorescent RNA detection probes to collect imagery from large number of cells to assess the number of CTCs. In this study, we spiked peripheral blood mononucleated cells (PBMCs) with SKBR-3 human breast cancer cells and added probes for relevant RNA targets such as EPCAM and Her-2. Taking advantage of the probes’ ability to detect RNA in live cells, combined with the capacity to acquire multi-spectral images of large numbers of cells, we demonstrate image based parameters that can be used to assess the frequency of CTCs in an objective and statistically significant manner. Citation Format: Shobana Vaidyanathan, Don Weldon, David Basiji, Philip Morrissey. Detection and enumeration of circulating tumor cells using imaging flow cytometery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3074. doi:10.1158/1538-7445.AM2014-3074