Objective To investigate the application of the TEM-1 beta-lactamase protein fragment complementation assay (PCA) in detecting weak and unstable protein-protein interactions as typically observed during chaperone-assisted protein folding in the periplasm of Escherichia coli. Results The TEM-1 beta-lactamase PCA system effectively captured the interactions of three pairs of chaperones and substrates. Moreover, the strength of the interactions can be quantitatively analyzed by comparing different levels of penicillin resistance, and the assay can be performed under 0.5% butanol, a stress condition thought to be physiologically relevant. Conclusions The beta-lactamase PCA system faithfully reports chaperone-substrate interactions in the bacterial cell envelope, and therefore this system has the potential to map the complex protein homeostasis network under a fluctuating environment.