A fully automated method for on-line ammonium determination in recycled water produced on board the International Space Station was developed and optimized. A trade off methodology is presented to evaluate and select the most appropriate analytical system, adaptable to space flight requirements. A sequential injection system meets the specifications for on-line monitoring in space conditions as it is an integrated flow manifold with small dimensions operating in a totally closed loop preventing any contact or release of the fluids/gases inside the space environment. The method is based on the reaction between ammonia and o-phthaldialdehyde in the presence of sulfite in alkaline media (pH ca. 11). The fluorescent product (isoindol-1-sulfonate) is then quantified at 425nm. The influence of chemical and flow variables as well as space adaptation characteristics which affect the performance of the system if placed on board the International Space Station have been studied, providing the appropriate conditions for the analysis of real samples. For a total analysis time of 174s, a detection limit (3s) of 0.018mgL−1 for NH4+ was obtained along with a sampling frequency of 20h−1. The working range was at values between 0.06 and 4.00mgL−1 NH4+. The “intra-day precision” was 2.30% (at 0.50mgL−1), while the “inter-day precision” was 2.40% (at 0.50mgL−1). The accuracy of the proposed method was evaluated by analyzing standard reference materials as well as using the certified method (indophenol blue) for ammonium determination. The method was successfully applied for ammonium determination in water samples obtained by the Water Treatment Unit Breadboard (WTUB) located in Antarctica Concordia Research Station.