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When living organisms are exposed to the space environment, numerous changes occur that affect virtually all cellular functions and molecular pathways. One class of understudied molecules in space biomedicine is noncoding RNAs (ncRNAs), particularly microRNAs (miRNAs). Given that spaceflight induces systemic health risks, miRNAs represent natural candidates for key regulators in space, as they can control hundreds to thousands of genes and influence central biological pathways. Transcriptomic data and pathway analyses are employed to identify the key genes driving specific physiological states, which can in turn be used to predict the miRNAs regulating those states. MiRNAs that act as upstream regulators of these driver genes are selected as candidates for a spaceflight-associated miRNA signature. Once these candidate miRNAs are identified, they must be experimentally validated. Depending on the health risk or biological process under investigation, miRNAs are validated either in circulating fluids (e.g., serum, plasma, blood) or in tissue samples using droplet digital polymerase chain reaction (ddPCR). Compared to conventional PCR methods, ddPCR enables absolute quantification of miRNA copy numbers and overcomes several limitations associated with traditional miRNA expression assays. This approach provides an unbiased prediction of candidate miRNAs, followed by precise and specific verification of their expression profiles, and can be broadly applied to identify miRNA signatures associated with various diseases and physiological stressors. Once validated, miRNAs that are overexpressed in response to spaceflight can be targeted for inhibition as potential countermeasures to mitigate health risks. Harnessing the power of miRNAs not only deepens our understanding of space biology but also accelerates the development of targeted interventions, ultimately enabling safe and sustained human exploration of deep space.