Currently, the strategy for determination of ITER in-vessel tritium inventory envisages that at predetermined intervals, tritiated gases in all systems of fuel cycle will be transferred to the storage and delivery system (SDS) and tritium quantities measured by in-bed calorimetry. The isotope separation system (ISS) is the system used to separate hydrogen isotopes at the quality required to be stored in SDS, and is one of the systems with highest tritium inventory within the fuel cycle. Therefore, during tritium inventory procedure, ISS has to be ‘milked down’ of tritium, mainly as DT molecular species. Based on the dynamic modelling code TRIMO of the tritium content in the main sub-systems of ITER Fuel Cycle, the procedure for tritium extraction from ISS is presented and numerical examples given to assess the necessary time of transferring the tritium from ISS to SDS, and the residual amount of tritium in ISS after different milking scenarios. Consequently a fuel handling strategy during tritium inventory assessment in the ISS and SDS is described, with the constraint of mobilizable tritium inventory minimisation.