Human macrophage migration inhibitory factor (MIF) was first recognized as a lymphokine that prevents macrophage migration in vitro. However, subsequent studies have revealed that MIF also functions as a chemokine, recruiting monocytes, neutrophils, and lymphocytes to sites of inflammation. While the role of its homologue, D-dopachrome tautomerase (D-DT or MIF-2), is less well understood, it shares both unique and overlapping functions with MIF. Both MIF and D-DT exhibit tautomerase catalytic activity, with MIF’s tautomerase active site being associated with health, but also the pathogenesis of various inflammatory conditions, autoimmune diseases, and cancers. In contrast, the role of D-DT’s tautomerase active site in disease remains poorly understood. In this study, we present a method for evaluating the tautomerase activity and inhibition of recombinant purified MIF and D-DT, using 4-hydroxyphenylpyruvate (4-HPP) and phenylpyruvate (PP) as substrates. Although both substrates can be used to measure MIF tautomerase activity, 4-HPP is preferred, while PP is favored for D-DT, highlighting the distinct substrate specificities of the two enzymes.