Amoeboid motion is characteristic of cells spanning the entire animal kingdom and is an essential process in embryogenesis, immune cell function, neural development and cancer metastasis.  Therefore, the molecular biology of this process has been intensely investigated.  In marked contrast, the basic motile behavior of amoeboid cells, which provides the contextual framework for interpreting the results of genetic and biochemical studies, has received far less attention. We lack an accurate and quantitative 4D description of how an amoeboid cell moves across a substratum. To obtain such information, we developed 3D-DIAS (Dynamic Image Analysis System) software to reconstruct and motion analyze the basic motile behavior of single cells including amoebae of the model system Dictyostleium discoideum, human polymorphonuclear leukocytes (PMNs) and human T-cells. By quantitating in space and time parameters such as the extension and retraction of pseudopods/lamellipodia, changes in shape, anterior progress of the main cell body and uropod and contact of the ventral surface of the cell with the substratum, we derived a complex morphometric description of cell migration with a number of defining characteristics that are altered in certain mutations.