AbstractPost‐translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two‐hybrid system employing human protein kinases for the analyses of phospho‐tyrosine (pY)‐dependent PPIs in a direct experimental, large‐scale approach. We identified 292 mostly novel pY‐dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co‐immunoprecipitation experiments from mammalian cells. About one‐sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY‐PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY‐mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY‐dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY‐dependent PPIs dictate cellular cancer phenotypes.