One of the major impediments to the implementation of displacement chromatography for the purification of biomolecules is the need to collect fractions from the column effluent for time-consuming offline analysis. The ability to employ direct online monitoring of displacement chromatography would have significant implications for applications ranging from analytical to preparative bioseparations. To this end, a set of novel fluorescent displacers were rationally designed using known chemically selective displacers as a template. Fluorescent cores were functionalized with different charge moieties, creating a homologous library of displacers. These compounds were then tested on two protein pairs, alpha-chymotrypsinogen A/ribonuclease A and cytochrome c/lysozyme, using batch and column displacement experiments. Of the synthesized displacers, two were found to be highly selective while one was determined to be a high-affinity displacer. Column displacements using one of the selective displacers yielded complete separation of both protein pairs while facilitating direct online detection using UV and fluorescence detection. Saturation transfer difference NMR was also carried out to investigate the binding of the fluorescent displacers to proteins. The results indicated a selective binding between the selective displacers and alpha-chymotrypsinogen A, while no binding was observed for ribonuclease A, confirming that protein-displacer binding is responsible for the selectivity in these systems. This work demonstrates the utility of fluorescent displacers to enable online monitoring of displacer breakthroughs while also acting as efficient displacers for protein purification.