Identifying activated T lymphocytes and differentiating antigen-specific from bystander T cells is crucial for understanding adaptive immune responses. This study investigates the efficacy of activation-induced markers (AIMs) in distinguishing these cell populations. We measured the expression of commonly used AIMs (CD25, CD38, CD40L, CD69, CD137, HLA-DR, ICOS, and OX40) in an in vitro T-cell activation system and evaluated their sensitivity, specificity, and positive predictive value. We demonstrated that individual AIMs, while specific in detecting activated CD4