Identification of T cell epitopes is a critical, but often difficult step in studying T cell function and developing peptide-based vaccines and immunotherapies. Unlike antibodies that recognize free soluble antigens, T cell receptor (TCR) recognizes its epitope bound to major histocompatibility complex (MHC) expressed on antigen presenting cells (APCs). In addition, the examination of T cell epitope activity requires the use of professional APCs, which are difficult to isolate, expand, and maintain. To address these issues, we have developed a facile, accurate, and high-throughput method for T cell epitope mapping by screening antigen-derived peptide libraries in complex with MHC protein displayed on yeast cell surface. Here, we use hemagglutinin and influenza A virus X31/A/Aichi/68 as examples to describe the key steps in identification of CD4+ T cell epitopes from a single antigenic protein and the entire genome of a pathogen, respectively. Methods for single-chain peptide-MHC complex vector design, yeast surface display, peptide library generation in Escherichia coli, and functional screening in Saccharomyces cerevisiae are discussed.