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Insect pests are a major limiting factor to producing profitable soybean (Glycine max (L.) Merr.) in South Carolina. Production practices within the soybean industry have drastically evolved over the last few decades, but treatment thresholds for insect pests have stayed the same. Evaluating treatment thresholds for insect pests typically involves simulating injury because it offers a controlled and repeatable way to evaluate an injury–yield relationship. Simulating defoliation injury in soybean typically involves methods such as hand-plucking or cutting leaflets, but these methods are not truly representative of insect feeding injury. This study describes the design, development, and validation of a novel pneumatic leaf puncher created with a 3D printer and used to simulate insect defoliation injury in soybean. The device was engineered to deliver controlled, repeatable leaf tissue removal at varying target levels (5, 15, 30, and 40%) by using interchangeable punching plates. Simulated defoliation treatments were applied to mature leaves on soybean plants at the V6 growth stage in a greenhouse study. The leaf area removed was quantified using LeafByte, a mobile app designed for measuring leaf area, and confirmed against target values. Results showed a high level of correlation between intended and actual defoliation levels, with accuracy ≥ 90%. The pneumatic leaf puncher provides a potential standardized method for administering foliar damage and offers a reliable alternative to manual clipping or herbivory feeding trials in defoliation research. Ongoing field trials at Clemson University will incorporate yield data to refine defoliation thresholds. Due to its adaptability and ease of use, the pneumatic leaf puncher could be implemented regionally, nationally, or internationally to support standardized defoliation studies across diverse cropping systems.