The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and tumor development. The hybrid MHC I complexed with heterologous β₂-microglobulin (β₂m) substitution from different species can be stabilized in vitro. This is a feasible means to study MHC I of mammals, when the homologous β₂m is not available. Meanwhile, it is indicated that mammalian β₂m substitution does not significantly affect peptide presentation. However, there is limited summarization regarding the methodology and the technology for the hybrid MHC I complexed with heterologous β₂-microglobulin (β₂m). Herein, methods to evaluate the feasibility of heterologous β₂m substitution in MHC I study are presented. These methods include preparation of expression constructs; purification of inclusion bodies and refolding of the MHC complex; determination of protein thermostability; crystal screening and structure determination. This study provides a recommendation for understanding function and structure of MHC I, and is also significant for T cell response evaluation during infectious disease and tumor immunotherapy.