Affinity chromatography with the subsequent identification of selectively binding proteins can be an efficient way to detect biologically significant molecular interactions. To optimize this method, it is necessary to take into account the structure of the molecule and its surface charge, as well as the possibilities to control its conformational changes, which can affect the hydrophobic properties of the surface. Using this approach, we identified several new target proteins of S100A4, including cytoskeleton proteins Sept2, Sept7, Sept11, and the transcriptional cofactor Ddx5. Interaction with septins may explains how S100A4 can affect cell motility, while its complex formation with Ddx5 apparently regulates the expression of E-cadherin, p21(Waf1/Cip1), Bnip3, and other genes. The proposed protocol can be applied to search for target proteins of other S100 family members, since their amino aside sequences and spatial structures are highly homologous.