We screened inhibitors in the adenylyl cyclase/protein kinase A/cAMP response element binding protein pathway (AC/PKA/CREB pathway) from a 2400 chemical library by a cell-based assay method using bioluminescence probes. We found a compound that inhibited forskolin-induced cAMP response element (CRE)-dependent transcription, the interaction between the kinase-inducible domain (KID) and the interacting domain (KIX), and endogenous CREB phosphorylation. Furthermore, this compound suppressed the activity of the PKA catalytic subunit dose-dependently. On the other hand, this compound did not inhibit forskolin-induced cAMP up-regulation. Taken together, we conclude that we have identified a new PKA inhibitor that binds to the catalytic subunit directly. We also succeeded in shortening the screening protocol by excluding a screening step which was used in a previous method.