Protein crystallography is a key technique of structural biology that allows researchers to determine the three-dimensional structure of proteins, protein–protein, protein–ligand, and protein–nucleic acid complexes at the atomic level. Whereas obtaining high-quality crystals is the first milestone for the structural characterization of a protein, the establishment of a detailed crystallization protocol promotes reproducibility of the crystallization process. Here, we describe a versatile crystallization method for human D-dopachrome tautomerase (D-DT or MIF-2), a small pleotropic protein of increasing interest due to its key role in human pathophysiology. The protocol described here provides a step-by-step procedure for generating high-quality protein crystals for a wide range of D-DT variants, including aggressive truncations and mutants with mechanistic value.