Inositol 1,4,5-trisphosphate (IP(3)) as a second messenger regulates complicated signaling processes in various physiological events. Development of a high-throughput screening system to monitor temporal changes of IP(3) is essential for screening of new potential therapeutic compounds. A simple, sensitive and rapid method for measuring IP(3) are described based on luciferase fragment assisted complementation strategy, which converts the ligand-induced conformational changes to light. Designed sensor comprising the IP(3)-binding core domain of IP(3)-receptor fused between complementary non-functional fragments of firefly luciferase allows direct detection of IP(3) in presence of luciferin substrate both in cell lysate and in living cells. Based on our design, the screening time was very fast and maximum response was obtained up to 11-fold higher than untreated cells. Moreover, the designed method was able to monitor release of IP(3) upon induction by Bradykinin. The current sensor not only provides a specific IP(3) detector in vitro but also facilitates monitoring of the response of IP(3) in living organisms.