The rapid development of proteomic technologies, combined with the completion of the Human Genome Map, has enabled the compiling of an unprecedented inventory of cellular proteins. Functional proteomics is an emerging field that aims to utilize the enormous amount of information provided by these proteomic technologies to understand the functions of cellular proteins. The utility of functional proteomics has been recently exploited to elucidate cellular mechanisms in numerous fields, of particular salience in the area of signal transduction. This review presents a functional proteomic approach for the study of cardiac cell signaling. It illustrates the strategies by which the subproteome of a targeted signaling system is characterized in an unbiased fashion, the manner in which the biochemical functions of this subproteome are assessed using established molecular and protein chemistry methods, and the challenges associated with these studies.