The Martian atmospheric Ne may reflect recent gas supply from its mantle via volcanic degassing, due to its short (∼100 Myr) escape timescale. The isotopic ratio of the Martian atmospheric Ne would therefore provide insights into that of the Martian mantle, further suggesting the origin of Mars volatiles during planetary formation. Mass spectrometric analysis of the Martian atmospheric Ne, however, has faced challenges from interference between 20Ne+ and 40Ar++. Previous studies using a polyimide membrane for 20Ne/40Ar separation were limited by the drawbacks of elastomeric O-rings to support the membrane, such as low-temperature intolerance, outgassing, and the need to endure environmental conditions during the launch and before/after landing on Mars. This study proposes a new method employing a metal C-ring to secure a 100 μm polyimide sheet within vacuum flanges. Environmental tests, including vibration, shock, extreme temperatures, and radiation exposure, were conducted on the gas separation flanges. Pre- and post-test analyses for He, Ne, and Ar demonstrated the membrane-flange system’s resilience. Gas permeation measurements using terrestrial air effectively permeated 4He and 20Ne, while reducing 40Ar by more than six orders of magnitude. This study achieved a <3% accuracy in determining the 20Ne/22Ne ratio, sufficient for assessing the origins of Ne in the Martian mantle. Furthermore, experiments with a 590 Pa gas mixture simulating the Martian atmosphere achieved a 10% accuracy for the 20Ne/22Ne isotope ratio, with gas abundances consistent with numerical predictions based on individual partial pressures. These results validate the suitability of the developed polyimide membrane assembly for in situ Martian Ne analyses.