Aim: Currently, therapeutic drug monitoring for astronauts faces limitations in conventional blood sampling and sample management onboard the international space station. Here, we explore the feasibility of dried blood spot (DBS) collection method during parabolic flights (PF) to overcome these constraints. Methods: We assessed the feasibility of blood deposition on blotting paper for preanalytical aspects in a PF using synthetic blood. Subsequently, DBS sampling validation was carried out in another PF campaign. Twenty volunteers participated in a pharmacokinetic study on caffeine and its metabolite, paraxanthine (as proof of concept), conducted during parabolic flights. After >18h caffeine washout, coffee (115 mg) or tea (30 mg), or 3 dark chocolate squares (11 mg) were administered. DBS samples were collected at baseline, during weightlessness, and post-flight. Caffeine and paraxanthine were analyzed using liquid chromatography-tandem mass spectrometry. Genotyping for Cytochrome P450-1A2 (CYP1A2) was performed and a metabolic ratio by areas under the curves for caffeine and paraxanthine (AUCPAX/AUCCAF) for CYP1A2 was determined. User experience survey was also conducted. Results: Full in-flight pharmacokinetic was feasible in 17 volunteers with 3 failures due to motion-sickness. We observed expected differences in kinetic profiles, consistent with consumption habits, the ingested dose and the genotypic/phenotypic information. The metabolic ratio did not significantly differ between parabolic flights conditions and ground conditions. Overall participants were satisfied with the usability of the method. Conclusion: DBS collection was safe, stable, feasible and well accepted in weightlessness. This method would offer valuable insights into human metabolism adaptation during long-term spaceflight, addressing space pharmacology challenges.