In the developing embryo, cell growth, differentiation and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here, we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immuno-affinity purified from zebrafish embryos at 3 and 5 days post fertilization and identified by multi-dimensional LC-MS. Amongst the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases and Focal adhesion kinases as well as the adaptor proteins, Paxillin, p130Cas and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immuno-affinity purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were observed. In addition, multiplex in vitro kinase assays were performed by incubating a micro-array of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation mediated signaling can be studied at endogenous levels in whole organisms.