The Golgi apparatus undergoes extensive disassembly during mitosis and reassembly in post-mitotic daughter cells. This process has been mimicked in vitro by treating Golgi membranes with mitotic and interphase cytosol. To determine the minimal machinery that controls the morphological change, we have developed a defined Golgi disassembly and reassembly assay that reconstitutes this process using purified proteins instead of cytosol. Treatment of Golgi membranes with mitotic kinases and COPI coat proteins efficiently disassembles the membranes into mitotic Golgi fragments, whereas further incubation with p97 or N-ethylmaleimide-sensitive factor (two AAA ATPases involved in membrane fusion) and their cofactors, in combination with protein phosphatase PP2A, leads to reassembly of the membranes into new Golgi stacks. The whole process takes 3–4 d and is applicable for identification and determination of novel cytosolic and membrane proteins that regulate Golgi membrane dynamics in the cell cycle.