An experimental methodology that facilitates functional analysis of numerous protein-protein interactions, which have been found in genome-wide interactome researches, has long been awaited. We propose herein an antagonistic inhibition-based approach. The antagonizing polypeptide is generated in the course of interaction domain mapping based on yeast 2-hybrid (Y2H) screening coupled with in vitro convergence of the Y2H-selected fragments, which is performed in a formatted procedure. Using the coupled methodology, we first performed a high-resolution mapping of an interdomain interaction network within budding yeast's Dam1 complex. Dam1 complex is a kinetochore protein complex composed of 10 essential subunits including Spc34p and Spc19p. The high-resolution mapping revealed the overall network structure within the complex for the first time: Dam1 components form into two separated subnetworks on N-terminal scaffolding domains of Spc34p and Spc19p, and the coiled-coil interaction in their C-terminal domains connects the subnetworks. Secondly, we show that the domain fragments converged in the high-resolution mapping acted as potent inhibitors for the endogenous interactions when episomally overexpressed. The in vivo Dam1 interaction targeting with the fragments conferred a similar phenotype on the host cells; a critical and irreversible damage, which was accompanied with disturbed budding and chromosome mis-segregation as a result of disorganized spindle. These phenotypes were strongly related to the cellular function of the Dam1 complex. The results and approach we demonstrated herein not only shed light on the Dam1 molecular architecture but also pave the road to reverse-interactome analysis and discoveries of novel drugs that target disease-related protein-protein interactions.