Tyrosine phosphorylation (pTyr)-dependent signaling pathways play vital role in various biological processes, which are spatiotemporally assembled and dynamically regulated on minute-scale by pTyr in living cells. Studying these pTyr-mediated signaling complexes is therefore challenging due to the highly dynamic nature of the protein complexes and the low abundance of pTyr. In this study, we adopted minute-resolution APEX2-based proximity labeling (PL) in living cells and Src SH2 superbinder-based pTyr peptide enrichment for simultaneously profiling these protein complexes and associated pTyr sites from the same affinity-purified sample. Upon different time-course of EGF stimulation of the living cells stably expressing APEX2-FLAG-GRB2, we constructed two-dimensional time-course curves for both interactome and tyrosine phosphoproteome.