Homeodomain transcription factors (HD TFs) are a large class of evolutionarily conserved DNA binding proteins that contain a basic 60-amino acid region required for binding to specific DNA sites. In Drosophila melanogaster, many of these HD TFs are expressed in the early embryo and control transcription of target genes in development through their interaction with cis-regulatory modules. Previous studies where some of the Drosophila HD TFs were purified required the use of strong denaturants (i.e. 6 M urea) and multiple chromatography columns, making the downstream biochemical examination of the isolated protein difficult. To circumvent these obstacles, we have developed a streamlined expression and purification protocol to produce large yields of Drosophila HD TFs. Using the HD TFs FUSHI-TARAZU (FTZ), ANTENNAPEDIA (ANTP), ABDOMINAL-A (ABD-A), ABDOMINAL-B (ABD-B), and ULTRABITHORAX (UBX) as examples, we demonstrate that our 3-day protocol involving the overexpression of His6-SUMO fusion constructs in E. coli followed by a Ni2+-IMAC, SUMO-tag cleavage with the SUMO protease Ulp1, and a heparin column purification produces pure, soluble protein in biological buffers around pH 7 in the absence of denaturants. Electrophoretic mobility shift assays (EMSA) confirm that the purified HD proteins are functional and nuclear magnetic resonance (NMR) spectra confirm that the purified HDs are well-folded. These purified HD TFs can be used in future biophysical experiments to structurally and biochemically characterize how and why these HD TFs bind to different DNA sequences and further probe how nucleotide differences contribute to TF-DNA specificity in the HD family.