We developed a revolutionary tricolor reporter in vitro assay system, in which three gene expressions are monitored simultaneously using green-, orange- and red-emitting beetle luciferases. The technique was used to analyze biological clock mechanisms, which are generated by complicated transcription-translation feedback loops of clock genes, and two clock gene transcriptions were monitored simultaneously. This technique, furthermore, could be utilized for fine analysis in new field of transcriptome and promoterome, as well as pharmacological or toxicological technologies.