Many critical protein–protein interactions are transient, making them challenging to study using established methods. This limitation is particularly evident in transport processes like bacterial secretion systems, where the interactions between the export machinery and the cargo proteins are inherently dynamic. In this protocol, we describe a sensitive method that allows for the identification of such interactions using the example of SycH, the chaperone of the bacterial type III secretion system (T3SS) effector YopH. Using this method in live Yersinia enterocolitica, we identified known as well as previously uncharacterized stable and transient interactors of this protein.