AbstractWe describe a simple and straightforward method for detection and characterization of X‐chromosome inactivation in females and/or individuals with more than one X chromosome. The X‐chromosome inactivation pattern is visualized on a single‐cell level using 5‐ethynyl‐2‐deoxyuridine (EdU) instead of the previously widely applied 5‐bromo‐2′‐deoxyuridine (BUdR). The fluorochrome‐labeled nucleoside analog EdU is incorporated into late‐replication chromosomal regions of living blood cells in vitro; thus, it can also be used to specifically highlight the inactive X chromosome within a cytogenetic preparation. The EdU‐based test for assessing skewed X‐chromosome inactivation can only be meaningfully applied if the X chromosome of the index patient can be cytogenetically distinguished under a microscope from the normal one. © 2018 by John Wiley & Sons, Inc.