AbstractNovel approaches to bio‐imaging and automated computational image processing allow the design of truly quantitative studies in developmental biology. Cell behavior, cell fate decisions, cell interactions during tissue morphogenesis, and gene expression dynamics can be analyzed in vivo for entire complex organisms and throughout embryonic development. We review state‐of‐the‐art technology for live imaging, focusing on fluorescence light microscopy techniques for system‐level investigations of animal development, and discuss computational approaches to image segmentation, cell tracking, automated data annotation, and biophysical modeling. We argue that the substantial increase in data complexity and size requires sophisticated new strategies to data analysis to exploit the enormous potential of these new resources. genesis 49:488–513, 2011. © 2010 Wiley‐Liss, Inc.