We developed a two-dimensional (2D) PEptide-centric Local Stability Assay (PELSA) which allows highly reliable target identification (~ 98% true identification for staurosporine) and systematic readout of multidimensional structural effects (direct interaction, allosteric regulation, complex regulation, etc.) induced by increasing ligand concentrations and local binding rate in native cellular environment. We then demonstrated the versatility of 2D-PELSA by investigating the interactions of 17β-estradiol (E2), a predominant sex hormone that exerts a wide spectrum function in both health and disease.