Based on N-cyclobutylaminoluciferin (cybLuc), a set of high and efficient caged bioluminescent derivatives (Clucs) as firefly luciferase pro-substrates has been developed herein. After careful examination, these molecules exhibited low cytotoxicity and prolonged bioluminescence imaging up to 6 h in vitro and in vivo. Importantly, these caged luciferin derivatives have the potential to serve as long-term tracking tools to explore some biological process by using bioluminescent imaging.