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Information regarding the physiology of African lions is scarce, mainly due to challenges associated with essential routine research procedures. The aim of this experiment was to test the possibility of training six captive lionesses by positive reinforcement conditioning (PRC) to voluntarily allow the collection of vaginal swabs and blood samples. This was done with the final goal of avoiding frequent anesthesia, and potentially stressful management during reproduction research. All lionesses mastered basic clicker and targeting principles within 2 weeks. Routine sampling was possible after 20 weeks of training, enabling the collection of about 750 vaginal swabs and 650 blood samples over 18 months. The animals remained calm and cooperative during all sessions, and demonstrated curiosity in the training. PRC training of captive lionesses proved to be a suitable, minimally invasive method for repeated collection of vaginal swabs and blood. Additionally, PRC may serve as behavioral enrichment for African lions in captive settings. Compared to chemical or physical restraining methods, this noninvasive management approach may reduce distress and physiological negative side effects, thus opening up new avenues for feline research.