We have developed several CARs targeting the cancer stem cell marker LGR5, one of which (CNA3103) is the lead candidate in an upcoming clinical trial in advanced metastatic colorectal cancer...This resulted in development of an assay that yielded very similar reproducibility and sensitivity to that previously observed with the indirect tEGFR surrogate assay. Overall, the findings highlight the utility of using CAR-targeting recombinant proteins for evaluating CAR expression on CAR-T cells, providing a general flow cytometry-based staining strategy that may be adapted for assessing a diverse repertoire of CARs.