Well-characterized silane layers are essential for optimized attachment of (bio)molecules enabling reliable chem/biosensor performance. Herein, binding properties and orientation of 3-mercaptopropyltrimethoxysilane layers at crystalline sapphire (0001) surfaces were determined by water contact angle measurements, infrared reflection absorption spectroscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Infrared reflection absorption spectroscopy measurements suggest an almost perpendicular arrangement of the MPTMS molecules to the substrate surface. Adhesion force studies between a silicon nitride AFM tip and modified sapphire, gold, and silicon dioxide substrates were investigated by peak force tapping atomic force microscopy and used to define the silane binding properties on these surfaces. As expected, the Al-O-Si bond was determined to be responsible for the layer formation at the sapphire substrate surface.