This work reports a new model substrate strategy to generate molecularly defined gradients of different affinity ligands on a surface that is compatible with cell culture conditions for cell adhesion, cell polarization and cell migration experiments. We demonstrate, for the first time, the subtle relationship between ligand slope, density and affinity on cell polarity and cell migration, a fundamental result in cell biology/materials and an area of intense research. Specifically, we show quantitatively that cells are able to modulate their migration position and behavior depending on the slope and density of immobilized peptide ligands. We synthesized two different peptide ligands (linear and cyclic RGD) that have different affinities for cell surface integrin receptors and show that cells are able to alter their migration requirements depending on the slope, affinity and density of the immobilized ligands. Because the surface is controlled and designed at the molecular level the only interaction between cell surface receptors and material is a ligand-receptor interaction, which is crucial for studying the complex events during cell motility. We also show single cell polarity on small gradient patterns is directed by the underlying peptide presenting gradient surface. Our surface chemistry strategy is general and can be used to immobilize a wide variety of ligands onto a surface with precise control of ligand slope and density for many other cell based assays, cell adhesion, polarization and migration experiments.