A Monte Carlo simulation was used to assess the performance of a collimated hollow X-ray microbeam for subcellular cytoplasm irradiation. A high-Z coaxial collimation structure with an inner core for nucleus shielding was investigated. Two key performances, the extraction efficiency (cytoplasm dose per unit incident fluence) and the dose contrast (cytoplasm-to-nucleus dose ratio), were evaluated regarding the influences of the material, geometry and physical arrangements of the collimator, target dish and incident beam source. Simulation results demonstrate that a gold coaxial structure with a practical collimation geometry of a 1-mm length, 10-μm inner diameter and 200-μm outer diameter, with the top exit closely attached (with a minimized air gap) to the bottom of a cell dish with a 3-μm thick Mylar film is recommended for cytoplasm irradiation of adherent mammalian cells. For a synchrotron source in the energy range < 10keV, a dose contrast of approximately 100 can be achieved. For a bremsstrahlung source <30-kV tube voltage, a dose contrast of approximately 50-100 can still be achieved. General principles are summarized with further explanations of the performance of the hollow X-ray microbeam.