Useful information is provided for determining the best startup sequence for multiple interlinked distillation columns for hydrogen isotope separation whose required output specifications are very strict. The column cascade developed for the Tritium Systems Test Assembly is chosen as an example. It is shown that the compositions of the gas mixtures charged into the columns have remarkable effects on the startup characteristics and should be carefully prepared. The compositions are determined by considering the inventories of hydrogen, deuterium, and tritium within the columns under full-normal (normal operating) conditions. Two strategies that are expected to present successful startup are found and discussed. One of the strategies is composed of only two operational modes, but has the complexity of charging four separate mixtures of different compositions into the columns. The other strategy avoids such complexity, but comprises seven modes and requires a roughly two times longer startup time. The control of the atomic fraction of tritium in the H2-HD stream conflicts with the purity control for the D2 stream. To assure the high purity of the D2 stream, the atomic fraction of tritium in the H2-HD stream must be decreased to an adequately low value before switching the operation to the full-normal mode.