Rhomboids (ROMs) constitute a family of polytopic serine proteases conserved throughout evolution. The obligate intracellular parasite Toxoplasma gondii possesses six genes coding for ROM-like proteases that are targeted to distinct subcellular compartments: TgROM1 localizes to regulated secretory organelles, micronemes, TgROM2 is present in the Golgi, while TgROM4 and TgROM5 are found in the pellicle of the parasite. The targeting mechanism/s of ROM proteins is an aspect that has not yet been assessed. The existence of TgROM family members localized to different subcellular compartments provides a convenient system to study their sorting mechanisms in a genetically tractable organism that possesses an elaborate secretory pathway and conserved trafficking machineries. In this study, we experimentally established the topology of TgROM1 and TgROM4 at the plasma membrane and applied domain-exchange and site-directed mutagenesis approaches to identify critical sorting determinants on the N-terminal cytosolic domains of TgROM2 and TgROM1 that confer their Golgi and post-Golgi localizations, respectively.