In our body cells move in three dimensions, embedded in an extracellular matrix that varies in composition, density and stiffness, and this movement is fundamental to life. Next to 3D cell migration assays, representing these physiological circumstances, still we need 2D migrations assays to perform detailed studies on the contribution of matrix-components and (extra) cellular proteins to cell movements. Next to the debate on differences between 3D and 2D migration, there also are many new perspectives on the use and development of novel or modified 2D cell migration assays. Of special significance is the introduction of so-called barrier migration assays, methods that avoid cell and matrix damage, as complementation or replacement of scratch/wound healing assays. Here, we discuss the possibilities and limitations of different 2D barrier migration assays. J. Cell. Physiol. 226: 288-290, 2010. (C) 2010 Wiley-Liss, Inc.