SUMO proteins are post-translational modifiers of the ubiquitin family, which are conjugated to thousands of target proteins, modifying their function or fate. SUMOylation involves a SUMO-E1 activating enzyme (Sae1/Sae2 heterodimer), a SUMO E2 conjugating enzyme (Ubc9), and, in some cases, SUMO E3s. Several deSUMOylases can remove the conjugated SUMO from the target, making SUMOylation an extremely dynamic process. The in vitro reconstitution of SUMOylation reaction using purified SUMO, E1, and E2 enzymes is a powerful tool for analyzing the effects of SUMOylation on specific target proteins, identifying novel regulators of SUMO conjugation/deconjugation, and screening for molecules that modulate this pathway. While these components are commercially available, their expression in E. coli and subsequent purification offers the possibility to produce large quantities, which can be required for high-throughput screening, structural, or biophysical approaches. In this protocol, we present optimized procedures for the production and purification of SUMO and the SUMO E1 and E2 enzymes, as well as an assay to control their activity.