Precise characterization of a monolayer of two different biomolecules in a gradient pattern on a glass surface puts high demand on the method used. Some techniques can detect protein monolayers but not on a glass surface. Others can distinguish between different proteins but not identify a gradient pattern. Here, we used ToF-SIMS to validate the complete surface composition, checking all the necessary boxes. As these types of surfaces can dictate sensitive cell behaviors, the precision on a nanolevel is crucial, and to visualize and determine the molecular distribution become essential. The dual monolayer consisted of laminin 521 and one of three other biomolecules of different sizes, epidermal growth factor, growth differentiation factor 5, or bovine serum albumin, creating opposing gradient patterns. The resulting ToF-SIMS imaging and line scan data provided detailed information on the distribution of the adsorbed proteins.