We have developed a novel gradient fabrication method for combinatorial surface studies that provides the equivalent of 5000 individual polyelectrolyte multilayer (PEM) film physicochemical conditions in a single 7 cm square film. A simple, inexpensive, and versatile automated layering instrument was built, which can generate a gradient of physical properties on a film in 1 dimension laterally by simultaneously changing both the location of polyelectrolyte adsorption and the layering conditions, such as pH or salt concentration of the polyelectrolyte dipping solutions. By rotating the substrate 90 degrees after each deposition cycle, full 2-dimensional gradient combinatorial films were fabricated over many layers, spanning virtually all previous combinations of stable deposition pH and salt conditions for both poly(allylamine hydrochloride) (PAN) and poly(acrylic acid) (PAA), a process which previously required more than 10 000 separate film samples. Surface spatial profiles energy (wettability), density (refractive index), and stiffness (modulus) were generated and correlated to assembly conditions. Additionally, step gradient films were generated first by varying the number of bilayers along one axis and pH along the other, which enabled us to measure their combined effect on thickness. To test for biocompatibility, we incubated HEK 293 cells on step gradient films and 2D combinatorial films for 48 h and determined that film assembly conditions played a major role, especially in controlling the stiffness and the density, which could be tailored with deposition pH over a wide range. Optimal growth conditions were discovered not at the extremes of fabrication pH, but instead near PAN pHs of 4-6 and PAA pH around 4, demonstrating that these PEM biosurfaces and this technique are suitable for optimizing high-throughput cellular screening. of film thickness, surface