The luciferase reporter system is useful for the assessment of various biological processes in vivo. The transcription factor pancreatic and duodenal homeobox 1 (Pdx1) is critical for the formation and the function of pancreatic β-cells. A novel reporter system using secreted Gaussia princeps luciferase (GLuc) under the control of a Pdx1 promoter was generated and activated in rat and mouse β-cell lines. This Pdx1-GLuc construct was used as a transgene for the generation of reporter mice to monitor Pdx1 promoter activity in vivo via the measurement of secreted GLuc activity in a small aliquot of blood. Significantly increased plasma GLuc activity was observed in Pdx1-GLuc mice. Analysis of Pdx1-GLuc mice by bioluminescence imaging, GLuc reporter assays using homogenates of various organs, and immunohistochemistry revealed that GLuc expression and activity were exponentially higher in pancreatic β-cells than in pancreatic non-β-cells, the duodenum, and other organs. In addition, GLuc activity secreted into the culture medium from islets isolated from Pdx1-GLuc mice correlated with the number of islets. The transplantation of Pdx1-GLuc islets into severe combined immunodeficiency mice elevated their plasma GLuc activity. Conversely, a partial pancreatectomy in Pdx1-GLuc mice reduced plasma GLuc activity. These results suggest that a secreted luciferase reporter system in vivo enables not only the monitoring of promoter activity but also a quantitative and minimally invasive assessment of physiological and pathological changes in small cell masses, such as pancreatic β-cells.