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Summary: Identifying defined T cell clones within a polyclonal population is key to clarifying their phenotype and function. Here, we present a protocol for detecting specified T cell clones in a heterogeneous cell population. We describe steps for stimulating human CD4+ T cells isolated from blood with a protein antigen, sorting antigen-specific cells by fluorescence-activated cell sorting, and detecting among these the presence of predefined T cell clones, based on their T cell receptor (TCR). TCR cDNA is amplified through 5′-RACE (TCR-SMART) and detected by qPCR.For complete details on the use and execution of this protocol, please refer to Notarbartolo et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.