Optical imaging of protein–protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. The present protocol demonstrates an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. A unique design of single-chain probes was fabricated, in which a full-length artificial luciferase (ALuc ®) was sandwiched between two model proteins of interest, e.g.,FKBP and FRB. A molecular tension probe comprising ALuc23 greatly enhances the bioluminescence in response to varying concentrations of rapamycin, and named “tension probe (TP)”. The basic probe design can be further modified towards eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named “combinational probe”. TPs may become an important addition to the tool box of bioassays in the determination of protein dynamics of interest in mammalian cells.