The desire to map reliable phosphorylation signaling network has motivated the development of high-performance techniques. Targeted biochemical studies and updated methods employing MS techniques are most used in mapping the phosphorylation sites and verifying novel interactions of kinases. Previously, we have established a novel method to efficiently facilitate more comprehensive, accurate phosphorylation site mapping of individual phosphoproteins by using combination of multiple stage MS analysis with target-decoy database search against the much smaller targeted database. In this study, by applying this method, we have identified the phosphorylation sites in human MSK1 mitogen- and stress-activated protein kinase 1), which has been proved to be a multi-phosphorylated kinase that plays key roles in various cell functions, activated by a novel interaction with MRK-beta. The results show that this method can find out not only those previously identified active sites in MSK1, but also some novel phosphorylated sites, which correlates with biochemical evidence that, besides p38 and extracellular signal-regulated kinase, MRK-beta could also activate MSK1 through direct interaction. Hence, we conclude this method is sensitive and reliable as expected and it can be further combined with automated screening and biochemical study in efficiently building up a more comprehensive phosphoprotein network.