We have established a in vivo limiting dilution assay (LDA) using the chick embryo chorioallantoic membrane (CAM), using a metastatic breast cancer cell model previously shown to be enriched for cancer stem cell (CSC) properties. This new tool was able to reproduce the same results found in mice, which is the gold standard method to evaluate CSC enrichment. Importantly, the chick model has several advantages, namely its easy handling, accessibility, rapid growth, lower costs and the absence of ethical and regulatory constraints.