We chose to use recently described fragments of Renilla reniformis luciferase for our assay because the light-emitting substrate of Rluc, coelenterazine, can easily pass through the cell envelope of Gramnegative bacteria, unlike the more common luciferase substrate D-luciferin, which requires acidic pH for optimal membrane permeability. As a first application for proof of concept, we fused these fragments to two chemotaxis proteins from Vibrio cholerae, the curved Gram-negative bacterium that causes human cholera. Classically, bacterial chemotaxis reflects the sensing of external stimuli by membrane-associated receptors that transmit a signal through a cytoplasmic cascade that modulates flagellar rotation. Membrane-embedded methyl-accepting chemotaxis proteins initiate chemotactic signaling upon detecting a change in local NVP-BEZ235 chemical gradients. Binding of a chemorepellent to its cognate MCP induces a conformational change that leads to autophosphorylation of a cytosolic kinase, CheA. CheA subsequently phosphorylates the response regulator CheY, which diffuses across the cell and binds the flagellar motor switch protein FliM. This binding switches the direction of flagellar rotation, which induces random reorientation of the cell. The lifetime of phosphorylated CheY is tightly regulated by the phosphatase CheZ, which hydrolyzes CheY’s phosphate group, thereby terminating the chemotactic signal. Pathway activity is also regulated by methyltransferase and methylesterase proteins, which modulate pathway sensitivity. Collectively, these proteins allow bacteria to maintain their direction under favorable conditions, and alter their direction under adverse conditions. V. cholerae encodes an unusually large number of putative MCPs, as well as three potentially independent clusters of downstream signaling proteins. Several cluster II genes, such as cheY3 and cheA2, are required for chemotaxis in vitro; however, genes from the other two clusters remain largely uncharacterized, and ligands have not been identified for any of the receptors. Progress in studies of bacterial chemotaxis has been hindered by a dependence on low-throughput and/or semi-quantitative chemotaxis assays. An assay based on the split luciferase technology could provide a sensitive, quantitative, and rapid microplate-based approach to studying bacterial chemotaxis, with particular utility for characterizing novel chemoeffectors. Importantly, elegant BRET and FRET analyses of chemotaxis by Berg and colleagues have demonstrated that the interaction of CheY and CheZ proteins in E. coli is directly proportional to chemotaxis pathway activity. However, due to their relative technical complexity, these assays are not widely used or easily adapted to a high-throughput format. We hypothesized that fusion of Rluc fragments to homologous proteins from V. cholerae would enable a direct measure of chemotactic responses and provide a more tractable platform for chemoeffector characterization. Here, we demonstrate that an Rluc-based PCA can be used to measure CheY3-CheZ interactions in V. cholerae and that this approach can quantify differences in chemotactic signaling. However, nonspecific inhibition of Rluc activity by small molecule effectors compromises the utility of this technique in measuring dynamic protein-protein interactions. These findings uncover a critical limitation of split luciferase complementation that may have broad implications for existing and future applications of this technology. These findings demonstrate the need for great caution in interpreting chemical effects on dynamic protein-protein interactions using split Rluc complementation.