Using a comparative phosphoproteomics approach, phosphotyrosine containing peptides of NS and LS-Shp2 expressing zebrafish embryos were isolated, identified and compared to peptides of WT embryos. A peptide corresponding to the autophosphorylation site of Fer K-Ras(G12C) inhibitor 9 kinase was identified as one of the most down-regulated peptides in developing NS and LS zebrafish. Further investigation revealed a role for Fer in C&E cell movements during gastrulation. Downregulation of Fer cooperated with NS and LS to induce developmental defects, suggesting a genetic interaction between Fer and the NS and LS variants of Shp2. Injection of both Fer morpholinos Promethazine hydrochloride induced developmental defects including reduced body axis length, heart edema and craniofacial defects at dpf. These defects were similar to the phenotypes observed in zebrafish expressing NS and LS Shp2 that were analyzed in parallel. MOs are known to induce non-specific p53 activation and apoptosis. To rule out that the observed phenotype is the mere result of p53 activation, co-injection of p53 MO was performed, which is an accepted control to assess specificity of MOs. Knockdown of p53 did not affect the fer knockdown phenotype, indicating that the phenotype was independent of p53. Taken together, two independent Fer MOs were used that blocked normal splicing of fer and induced developmental defects. Using a comparative phosphoproteomics approach focused on pTyr-containing proteins, we identified a phosphopeptide corresponding to Fer kinase as the main decreased phosphopeptide in zebrafish embryos expressing NS or LS mutant Shp2 compared to WT. Fer knockdown induced developmental defects in zebrafish embryos that are reminiscent of defects induced by NS and LS Shp2. Epistatic interaction analyses suggested a genetic interaction between Fer kinase and mutant Shp2. Previously, we used comparative phosphoproteomics in zebrafish to identify differences between wild type and Fyn/Yes knockdown embryos. At that time, TiO2 columns were used to enrich for phosphopeptides, and only highly abundant phosphoserine and phosphothreonine-containing phosphopeptides were identified, excluding low abundant pTyr-containing peptides.