Peptide microarrays are a fast-developing field enabling the mapping of linear epitopes in the immune response to vaccinations or diseases and high throughput studying of protein-protein interactions. In this respect, a rapid label-free measurement of protein layer topographies in the array format is of great interest but is also a great challenge due to the extremely low aspect ratios of the peptide spots. We have demonstrated the potential of vertical scanning interferometry (VSI) for a detailed morphological analysis of peptide arrays and binding antibodies. The VSI technique is shown to scan an array area of 5.1 square millimeters within 3–4 min at a resolution of 1.4 μm lateral and 0.1 nm vertical in the full automation mode. Topographies obtained by VSI do match the one obtained by AFM measurements, demonstrating the accuracy of the technique. A detailed topology of peptide-antibody layers on single spots was measured. Two different measurement regions are distinguished according to the antibody concentration. In the case of weakly diluted serum, the thickness of the antibody layer is independent of the serum dilution and corresponds to the physical thickness of the accumulated antibody layer. ... mehrIn strongly diluted serum, the thickness measured via VSI is linearly proportional to the serum dilution.