TCR-T cell therapy holds great promise for solid tumour treatment, but its feasibility relies critically on identifying antigen-specific T cell receptors (TCRs) capable of recognizing cancer cells. This typically involves characterising tumour-infiltrating lymphocytes (TILs); however, the majority of TILs are bystander cells, making the identification of antigen-specific tumour-reactive T cells a laborious and costly "needle in a haystack" problem. Hence, we aim to establish and validate a gene-signature based approach to rapidly identify antigen-specific T cells based solely on their transcriptional profiles. We stimulated peripheral blood mononuclear cells (PBMCs) in vitro with peptide pools derived from SARS-CoV-2, Influenza A (IAV), and Epstein-Barr Virus (EBV) and sorted total activated T cells, encompassing both TCR- and bystander- activated populations, for single-cell RNA (scRNA-seq) and T cell receptor sequencing (scTCR-seq). In parallel, PBMCs were stained with the corresponding MHC class I multimers to identify known CoV-2/IAV/EBV- specific TCR clonotypes. These clonotypes information was used to annotate the ground-truth TCR- and bystander-activated T cells within the peptide-activated T cells. We subsequently applied a TCR activation–associated gene signature derived from a public dataset to these two populations and validated the signature’s ability to robustly distinguish TCR-activated T cells from bystanders. As a proof of concept, TCRs from ground-truth TCR-activated T cells were reconstructed from scTCR-seq data and electroporated into autologous T cells. These TCR-engineered T cells responded specifically to stimulation with their cognate peptide pools, confirming their antigen specificity. Applying this validated signature on the remaining peptide-activated T cells, we shortlisted putative antigen-specific T cells and future studies will functionally validate these signature-derived candidates. In conclusion, we show that it is feasible to use a transcriptional signature-based approach to rapidly discriminate bona fide TCR-activated antigen-specific T cells from cytokine-activated bystander T cells, providing a scalable strategy for TCR discovery in TCR-T immunotherapy.