Besides the most common glass substrates, novel materials are pursued as substrates for protein microarrays, including flat polymer substrates and porous substrates, such as nitrocellulose, porous silicon and synthetic paper. Microstructures on a substrate surface affect the spot properties, and thereby the assay performance. We investigated the influence of substrate microstructures on the properties of microarray spots. We run a noncompetitive immunochemical assay on glass, nitrocellulose and variants of synthetic paper. We compare the fluorescent intensity profile, size, roundness, and coffee ring ratio of spots on different substrates. We found that spot size is related to the pore size, porosity and volume capacity of the microstructures. Microstructures can reduce the roundness and coffee ring ratio of the microarray spots.