Adeno-associated virus (AAV) is showing promise as a therapy for diseases that contain a single-gene deletion or mutation. One major scale-up challenge is the removal of empty or non-gene of interest containing AAV capsids. Analytically, the empty capsids can be separated from full capsids using anion exchange chromatography. However, when scaled up to manufacturing, the minute changes in conductivity are difficult to consistently obtain. To better understand the differences in the empty and full AAV capsids, we have developed a single-particle atomic force microscopy (AFM) method to measure the differences in the charge and hydrophobicity of AAV capsids at the single-particle level. The atomic force microscope tip was functionalized with either a charged or a hydrophobic molecule, and the adhesion force between the functionalized atomic force microscope tip and the virus was measured. We measured a change in the charge and hydrophobicity between empty and full AAV2 and AAV8 capsids. The charge and hydrophobicity differences between AAV2 and AAV8 are related to the distribution of charge on the surface and not the total charge. We propose that the presence of nucleic acids inside the capsid causes minor but measurable changes in the capsid structure that lead to measurable surface changes in charge and hydrophobicity.