The ability of resupplying fluid is vital to many aerospace applications for the sake of lifetime prolongation and performance maintenance, especially for deep-space explorations. Compared to conventional on-orbit filling technologies with the emphasis on efficiency maximization, fluid replenishment for two-phase cooling systems in space requires an accurate control of the transferred quantity constrained by its working principle. Under microgravity, weighing is impractical, and due to the dependent relationship between the saturated temperature and the saturated pressure, mass cannot be deduced from measurements of the two thermal properties, making the filled quantity determination challenging in space. For this reason, this article presents a novel quantitative on-orbit filling method for two-phase systems, through which the transferred mass is obtained from the density variation of the supercritical fluid in the supply vessel. Via heating the fluid to supercritical, a homogeneous state independent from gravity is achieved, and therefore the fluid density can be deducted from the temperature and the pressure. The filling process is driven by pressure difference and precisely regulated by solenoid valves and a flow restrictor situated in the transfer line, constituting two fundamental components. To direct the filling, an empirical model aiming to predict the transferred mass is established in terms of the pressure difference and transfer time. Based on that, a fill/refill architecture is deployed to transfer a quantitative amount of coolant to a delicate pumped CO2 two-phase thermal control system, the Upgraded Tracker Thermal Pump System in the Alpha Magnetic Spectrometer-02 on the International Space Station, which was designed and constructed to replace the current Tracker cooling system in 2019. Results of the test conducted in a thermal-vacuum chamber showed that the density-variation approach in the upgraded two-phase system achieved a filling accuracy better than 7%, and the empirical model showed consistency with the approach within 5%. Moreover, a criterion to assess when the two-phase cooling system needs a coolant refill was proposed according to the investigation of a two-phase fluid accumulator of the system under thermal regulation, in which the heat transfer is strongly affected by liquid-vapor distribution. Given the above, the proposed method contributes to the on-orbit filling for two-phase systems with more precise control over the transferred quantity. It also delivers valuable insights to the operational status evaluation of two-phase systems for future space missions.