The autonomous migration of specialized cells is an essential characteristic in both physiological and pathological functions in the adult human organism. Leukocytes, fibroblasts, and stem cells, but also tumor cells, are thus the subject of intense investigation in a broad range of research fields. A wide spectrum of methods have therefore been established to analyze chemokinetic and chemotactic cell migration, ranging from easy-to-handle two-dimensional surface migration assays to highly specialized three-dimensional and intravital analysis methods. It is now manifest that the results obtained with these various migration assays substantially differ. This review therefore gives an overview of the migration assays which are currently in use, describes the methods, and critically enlightens the particular advantages and disadvantages of each method.