Background: Enhancing the motilities of stem cells has been of a great concern in the applications of stem cells for repairing bodily organs. Boyden chamber test, typically employed in a motility test, only tells whether the number of transmigrated cells is greater or smaller compared to the number of a control. Western blotting also test only tells if a particular protein related to the motility is more abundantly expressed or less. Both tests do not reveal the detailed cell migration process. Phase retardation imaging, which is label free, can continuously reveal in real time the dynamic movement of cytoskeletal proteins in stem cells. Methods: The dynamic movement of the cytoskeletal proteins of UCB-MSCs, such as actins and tubulins, was imaged with a label free, phase retardation imaging technique. The fluorescent images of other molecules involved in the process of migration, such as integrin, were compared with the phase retardation images. Results: The phase retardation images vividly showed the dynamically changing cytoskeletal structures. Even though the cells were not moving macroscopically and their backbone skeleton remained unchanged, the cytoskeletal proteins in the focal adhesion points were constantly arranging and rearranging. Especially, after the cells were treated with a motility enhancing agent, they were much more active in lateral movement than before. The moving speed of cell periphery reached up to several tens of micrometers per hour. Importantly, the images clearly revealed how multiple focal adhesion points were coordinating beautifully one another. Microscopically, each focal adhesion point in the oxytocin treated cells continuously mobilized and reorganized cytoskeletal proteins much faster than before the treatment. The integrin rich areas corresponded to the focal adhesion points. Conclusion: Compared to Boyden chamber test and Western blotting, a phase retardation imaging technique showed dynamically the process of cell migration in detail. Even while the cells were not moving, the phase retardation images vividly showed how the focal adhesion points coordinate one another and that the cytoskeletal proteins in the focal adhesion points constantly changed their arrangement. Even though the abundance of a particular molecule related to the migration process could predict the motility of the cell, the efficiency of utilization of the proteins, such as assembly and disassembly of the proteins, might be more important in the migration process.