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Introduction: The future of human space missions relies on the ability to provide adequate food resources for astronauts and also to reduce stress due to the environment (microgravity and cosmic radiation). In this context, microgreens have been proposed for the astronaut diet because of their fast-growing time and their high levels of bioactive compounds and nutrients (vitamins, antioxidants, minerals, etc.), which are even higher than mature plants, and are usually consumed as ready-to-eat vegetables. Methods: Our study aimed to identify the best light recipe for the soilless cultivation of two cultivars of radish microgreens (Raphanus sativus, green daikon, and rioja improved) harvested eight days after sowing that could be used for space farming. The effects on plant metabolism of three different light emitting diodes (LED) light recipes (L1—20% red, 20% green, 60% blue; L2—40% red, 20% green, 40% blue; L3—60% red, 20% green, 20% blue) were tested on radish microgreens hydroponically grown. A fluorimetric-based technique was used for a real-time non-destructive screening to characterize plant methabolism. The adopted sensors allowed us to quantitatively estimate the fluorescence of flavonols, anthocyanins, and chlorophyll via specific indices verified by standardized spectrophotometric methods. To assess plant growth, morphometric parameters (fresh and dry weight, cotyledon area and weight, hypocotyl length) were analyzed. Results: We observed a statistically significant positive effect on biomass accumulation and productivity for both cultivars grown under the same light recipe (40% blue, 20% green, 40% red). We further investigated how the addition of UV and/or far-red LED lights could have a positive effect on plant metabolite accumulation (anthocyanins and flavonols). Discussion: These results can help design plant-based bioregenerative lifesupport systems for long-duration human space exploration, by integrating fluorescence-based non-destructive techniques to monitor the accumulation of metabolites with nutraceutical properties in soilless cultivated microgreens. [ABSTRACT FROM AUTHOR]