Laser Raman spectroscopy has been applied to the on-line analysis of the operation of the cryogenic Isotope Separation System (ISS) at the Tritium Systems Test Assembly (TSTA). A flow-through cell was employed to permit near real-time observation of the dynamic response of the 3-column ISS. Accurate analysis of hydrogen isotopic mixtures may be made in less than 2 minutes. Full response to a change in the sampling point is achieved in approximately one minute. Response measurements are shown as well as static column profiles and dynamic response to induced parameter changes. Cross check of analysis was performed with radio gas chromatography.