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Abstract Chemokines are important immune system proteins, many of which mediate inflammation due to their function to activate and cause chemotaxis of leukocytes. An important anti‐inflammatory strategy is therefore to bind and inhibit chemokines, which leads to the need for biophysical studies of chemokines as they bind various possible partners. Because a successful anti‐chemokine drug should bind at low concentrations, techniques such as fluorescence anisotropy that can provide nanomolar signal detection are required. To allow fluorescence experiments to be carried out on chemokines, a method is described for the production of fluorescently labeled chemokines. First, a fusion‐tagged chemokine is produced inEscherichia coli, then efficient cleavage of the N‐terminal fusion partner is carried out with lab‐produced enterokinase, followed by covalent modification with a fluorophore, mediated by the lab‐produced sortase enzyme. This overall process reduces the need for expensive commercial enzymatic reagents. Finally, we utilize the product, vMIP‐fluor, in binding studies with the chemokine binding protein vCCI, which has great potential as an anti‐inflammatory therapeutic, showing a binding constant for vCCI:vMIP‐fluor of 0.37 ± 0.006 nM. We also show how a single modified chemokine homolog (vMIP‐fluor) can be used in competition assays with other chemokines and we report aK_dfor vCCI:CCL17 of 14 μM. This work demonstrates an efficient method of production and fluorescent labeling of chemokines for study across a broad range of concentrations.