S100 proteins are small, mostly dimeric, EF-hand Ca
-binding proteins. Upon Ca
binding, a conformational change occurs resulting in the exposure of a shallow hydrophobic binding groove in each subunit. Interestingly, S100 proteins can interact with their partners in two ways: Symmetrically, when the two partners identically bind into each groove, or asymmetrically, when only one partner binds to the S100 dimer occupying both binding pockets. Here we present a heterologous expression and purification protocol for all known human S100 proteins as well as for their partner peptides. Moreover, we provide a detailed description of three in vitro methods to determine the affinity, stoichiometry, and kinetics of S100 protein-protein interactions.