In recent years the differentiation efficiency of human embryonic stem cells (hESCs) to cardiomyocytes has improved considerably. In general, hESC-derived cardiomyocytes are formed in aggregates, which require dissociation for follow-up experimental analyses and (clinical) applications. Here, we show that inhibition of the Rho-associated kinase (ROCK) by Y-27632 improved survival of dissociated hESC-derived differentiated cells. A maximum effect on cell survival was already observed within the first 24 hours. Hereafter, no further differences in the percentage of apoptotic and proliferating cells were observed with or without ROCK-inhibitor treatment. Improved survival was observed in both cardiomyocyte as well as non-cardiomyocyte cell populations. Viable cardiomyocytes were indicated by the appearance of beating, sarcomeric organization of cardiac-specific proteins, and fluorescence of a mitochondrion-selective dye. These results facilitate development of applications of hESC-derived cardiomyocytes in multiple research areas. Furthermore, these findings may be applied to other cell types differentiated from hESCs or other stem cells.