S-Layer proteins are an example of bionanostructures that can be exploited in nanofabrication. In addition to their ordered structure, the ability to self-assembly is a key feature that makes them a promising technological tool. Here, in vitro self-assembly kinetics of SpbA was investigated, and found that it occurs at a rate that is dependent on temperature, its concentration, and the concentration of calcium ions and sodium chloride. The activation enthalpy (120.81 kJ · mol−1) and entropy (129.34 J · mol−1 · K−1) obtained infers that the incorporation of monomers incurs in a net loss of hydrophobic surface. By understanding how the protein monomers drive the self-assembly at different conditions, the rational optimization of this process was feasible.