Death Associated Protein Kinase (DAPk) is a Ser/Thr kinase whose activity is necessary for different cell death phenotypes. While its contribution to cell death is well established, only a handful of direct substrates have been identified, which do not fully account for DAPk’s multiple cellular effects. In order to identify such substrates on a large scale, we developed an in vitro, unbiased, proteomics-based assay to search for novel DAPk substrates. Biochemical fractionation and mass spectrometric analysis was used to purify and identify several potential substrates from HeLa cell lysate. Here we report the identification of two such candidate substrates, the ribosomal protein L5 and Mcm3, a replication licensing factor. While L5 proved to be a weak substrate, Mcm3 was efficiently and specifically phosphorylated by DAPk on a unique site, Ser160. Significantly, DAPk phosphorylates this site in vivo upon over-expression in 293T cells. Activation of endogenous DAPk by increasing intracellular Ca²⁺ also leads to increased phosphorylation of Mcm3. Importantly, shRNA-mediated knock-down of endogenous DAPk blocks both basal phosphorylation and Ca²⁺-induced phosphorylation, indicating that DAPk is both necessary and sufficient for Mcm3 Ser160 phosphorylation in vivo. Identification of Mcm3 as an in vivo DAPk substrate indicates the usefulness of this approach for identification of physiologically relevant substrates that may shed light on novel functions of the kinase.