Insulin has so far been the most important pharmaceutical peptide for diabetes treatment, assisting to regulate carbohydrate and fat metabolism in patients. However, aggregation of insulin occurs readily in almost every biopharmaceutical process, ranging from production, purification, storage, transportation, delivery, to in vivo utilization at the terminal. As interfaces and surfaces are ubiquitous in each process and strongly influence physical/chemical properties of insulin, it is necessary and fundamentally important to investigate the aggregation of insulin at various interfaces, such as aqueous-solid interface, water-oil interface, and air-water interface. The objective of this article is to briefly summarize recent progress on insulin aggregation at different interfaces, with special focus on the air-water interface using the Langmuir monolayer technique.