The human insulin (HI) protein was examined to elucidate its structure at the air-water interface. Optimal experimental conditions were determined to prepare a homogeneous and stable human insulin (HI) Langmuir monolayer. HI insulin Langmuir monolayer can be used to study interactions of HI with a membrane as Langmuir monolayers are used as an in vitro model of biological membranes. Surface pressure and surface potential-area isotherms were used to characterize the HI Langmuir monolayer. The compression-decompression cycles and stability measurements showed a homogeneous and stable monolayer at the air-water interface. However, higher surface pressures resulted in a higher decrease in area and less stability. In situ UV-vis and fluorescence spectroscopy were used to verify the homogeneity of the HI monolayer and to identify the chromophore residues in the HI. Domain formation was examined through epifluorescence and Brewster angle microscopies. The conformation of HI was examined by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) in the aqueous phase and at the air-water interface by infrared reflection absorption spectroscopy (IRRAS). HI was found to exist as a monomer in 2-D.