Effector functions and cellular properties of natural killer (NK) cells are regulated by cellular and extracellular factors shaped by the microenvironments. NK cells express specific chemokine and non-chemokine receptors to aid preferential migrations or localizations in tissues. Good understanding of how NK-cell migratory properties are regulated in physiological and pathological microenvironments will provide further insights into the development of NK cell-based therapeutic approaches. In contrast to the commonly used conventional in vitro migration assays such as Trans-well assays that measure movements of a population of the migratory cells, microfluidic-based devices support live-cell imaging of cell migrations under a well-defined chemical gradient(s) at microscale. Subsequent analyses at single-cell level provide quantitative measurements of cell-migration parameters such as speed and Chemotactic Index, and permit distinguishing chemotaxis, chemokinesis, and chemo-repulsion. Our recent work established the use of a Y-shaped microfluidic device to study NK cell migrations in vitro. In this chapter, we described the detailed method of acquiring and analyzing NK cell migration in the microfluidic devices.