Abstract: We describe a computational protocol to identify functional modules and pathway relationship of chaperones based on physical interaction data derived from high-throughput proteomic experiments. The protocol first identifies interacting proteins shared by the different chaperone systems to organize the chaperones into functional modules. The chaperone functional modules represent groups of chaperones that are involved in mediating the folding of the shared interacting proteins. Either the chaperones in a module can function along a single folding pathway of a given substrate protein or the substrate protein might have two or more different folding pathways that the chaperones act on independently. As described in our computational protocol, probabilities of these pathway relationships between two chaperones in a two-component chaperone module can be determined using whole-genome expression and cellular pathways as reference. This protocol is potentially useful for identifying functional modules and pathway relationships in other biological systems that involve multiple proteins with many identified interactions.