Updated PubMed. Xeno-free culture systems have expanded the application of human pluripotent stem cells (PSCs). Here we describe an improved method for the subculture of human PSCs. The revised method significantly facilitated the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications. Furthermore, the method did not alter the PSC characteristics after long-term culture and attenuated the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and improves the quality control of human PSC research and applications.