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Summary: M1- and M2-like macrophages infected with Mycobacterium tuberculosis (Mtb) have been found to differ in their capacity to elicit memory CD4+ T cell activation. Here, we present a protocol to quantify and isolate the subset of human memory CD4+ T cells activated in response to autologous monocyte-derived macrophages (MDMs) infected with virulent Mtb. We describe steps for CD14+ monocyte isolation, generating MDMs, culturing Mtb and infection of macrophages, and identifying activated CD4+ T cells by flow cytometry.For complete details on the use and execution of this protocol, please refer to Gail et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.