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Summary: Here, we provide a protocol for the design, expression, purification, and functional studies of an engineered trimeric version of the receptor-binding domain (tRBD) of SARS-CoV-2 spike protein. We describe the use of tRBD to block SARS-CoV-2 spike pseudovirus and true virus binding to cellular angiotensin converting enzyme-2 (ACE2), thereby blocking viral infection. This protocol is applicable to generate a trimeric version of any protein of interest.For complete details on the use and execution of this protocol, please refer to Basavarajappa et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.