Two-component signal transduction systems, composed of histidine kinases and response regulators, enable bacteria to sense, respond, and adapt to changes in their internal and external conditions. The importance of these signaling systems is reflected in their widespread distribution and prevalence in the bacterial kingdom, with some organisms encoding as many as 250 two-component signaling proteins. In many cases, a histidine kinase and a response regulator are encoded in the same operon and, in such cases, the two molecules usually interact in an exclusive one-to-one fashion. However, in many organisms, the vast majority of two-component signaling genes are encoded as orphan genes, precluding the mapping of signaling pathways based on sequence information and genome position alone. There is also a growing number of examples of two-component signaling pathways with more complicated topologies, including one-to-many and many-to-one relationships, which cannot be inferred from sequence. To address these problems, we have developed an in vitro technique called phosphotransfer profiling, which enables the systematic identification of two-component signaling pathways. Purified histidine kinases are tested for their ability to transfer a phosphoryl group to each response regulator encoded in a genome of interest. As histidine kinases typically exhibit a strong kinetic preference in vitro for their in vivo cognate substrates, this technique allows the rapid mapping of cognate pairs and is applicable to any organism containing two-component signaling genes. The technique can be further extended to mapping phosphorelays and the cognate partners of histidine phosphotransferases. Here, we describe protocols and strategies for the successful implementation of this system-level technique.