Calcium dependent processes are often impaired in myocardial dysfunctions and calcium-binding proteins might be involved as mediators of Ca2+ signals. Here we present a method to assess a footprint of the calcium-binding proteins of the S100 family in human heart. The S100 proteins are purified through calcium dependent affinity chromatography and reverse phase high-performance liquid chromatography and are analyzed by electrospray-ionization mass spectrometry (ESI-MS) and liquid secondary ionization/tandem quadrupole mass spectrometry (LS-MS/MS). In human heart we identified S100 alpha, CACY, and CAPL as the most abundant S100 proteins and showed that they occur as monomers and homodimers.