Kinase-substrate networks are the main components of many signal transduction pathways. Although proteome-wide studies have been successful in elucidating many important biological events including the cataloging of thousands of sites of protein phosphorylation, there is a lack of a universal method to identify the direct upstream kinases responsible for many of these modifications. We have introduced Fluorescence ComplementatiKinase-substrate networks are the main components of many signal transduction pathways. Although proteome-wide studies have been successful in elucidating many important biological events including the cataloging of thousands of sites of protein phosphorylation, there is a lack of a universal method to identify the direct upstream kinases responsible for many of these modifications. We have introduced Fluorescence Complementation Mass Spectrometry (FCMS) as the first proteomic approach that identifies direct upstream kinases in living cells by stabilizing and capturing the kinase-substrate pairs. Using FCMS, we have identified both known and novel direct kinases of cAMP response element-binding protein (CREB). on Mass Spectrometry (FCMS) as the first proteomic approach that identifies direct upstream kinases in living cells by stabilizing and capturing the kinase-substrate pairs. Using FCMS, we have identified both known and novel direct kinases of cAMP response element-binding protein (CREB).