The recognition of defined antigen‐MHC complexes by antigen‐specific T cells forms the molecular basis of T cell immunity. It has been shown that fluorescently labeled recombinant MHC tetramers can be utilized to detect antigen‐specific T cells by flow cytometry. Since this first description, MHC tetramers and other types of MHC multimers have become a core tool to monitor the development of disease‐ and therapy‐induced antigen‐specific T cell responses both in humans and in animal model systems. This unit describes a set of protocols that transform classical MHC multimer technology into a high‐throughput platform, allowing one to produce large collections of MHC class I molecules charged with different peptides. This technology is based on the development of conditional MHC ligands that can be triggered to self‐destruct while in the MHC‐bound state. Curr. Protoc. Immunol. 87:18.16.1‐18.16.20. © 2009 by John Wiley & Sons, Inc.