<p>Cell migration is a key cellular process that drives major developmental programs. To mimic and mechanistically understand cell migration in these different contexts, different assays have been developed. However, owing to the lack of practical guidelines, these different cell migration assays are often used interchangeably. This and the inherent dynamic nature of cell migration, which often requires sophisticated live-cell microscopy, may have caused cell migration to be notably less well understood than equally important cell functions, such as cell differentiation or proliferation. In this Review, we describe commonly used custom and commercial in vitro and in vivo cell migration assays and provide a comprehensive practical guide and decision tree outlining how to choose and implement an assay that best suits the biological question at hand. We hope this guidance spurs biological insights into this complex process and encourages future methods development.</p>