Understanding and identifying protein–ligand interactions is essential for elucidating fundamental biological processes and developing translational applications such as vaccine and drug targets. Yeast surface display (YSD) systems expressing genome-wide or combinatorial libraries in Saccharomyces cerevisiae are powerful tools to identify protein–ligand interactions in an unbiased fashion. In the following protocol, we couple the YSD system expressing a genome-wide library of the protozoan pathogen Trypanosoma cruzi with magnetic-activated cell sorting to identify antigen–antibody interactions. We describe the enrichment of pathogen antigens targeted by antibodies of infected patients using the YSD library. We also detailed a DNA sequencing methodology and a data analysis computational pipeline. The approach can be easily adapted to identify protein–protein or protein–drug interactions.