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SUMMARY During organismal development, differential regulation of the cell cycle is critical to many cell biological processes, including cell fate specification and differentiation. While the mechanisms of cell cycle regulation are well studied, how control of the cell cycle is linked to differentiated cellular behavior remains poorly understood, mostly due to our inability to directly and precisely measure cell cycle state. In order to characterize cell cycle state live, we adapted a cyclin-dependent kinase (CDK) biosensor for in vivo use in the roundworm nematode, Caenorhabditis elegans . The CDK biosensor measures the cytoplasmic-to-nuclear localization of a portion of human DNA Helicase B (DHB) fused to a fluorescent protein to assess cell cycle state. The dynamic localization of DHB results from phosphorylation of the biosensor by CDKs, thereby allowing for quantitative assessment of cell cycle state. We demonstrate here the use of this biosensor to quantify lineage-specific differences between cycling cells and to examine the proliferation-differentiation decision. Unlike other live cell imaging tools (e.g., FUCCI), we show that DHB can be used to distinguish between actively cycling cells in the G 1 phase of the cell cycle and terminally differentiated cells exited in G 0 . Thus, we provide here a new resource to study the control and timing of the metazoan cell cycle during cell fate specification and differentiation.