Intracellular pathogens such asMycobacterium tuberculosis(Mtb) have evolved diverse strategies to counteract macrophage defence mechanisms including phagolysosomal biogenesis. Within macrophages, Mtb initially resides inside membrane‐bound phagosomes that interact with lysosomes and become acidified. The ability of Mtb to control and subvert the fusion between phagosomes and lysosomes plays a key role in the pathogenesis of tuberculosis. Therefore, understanding how pathogens interact with the endolysosomal network and cope with intracellular acidification is important to better understand the disease. Here, we describe in detail the use of fluorescence microscopy‐based approaches to investigate Mtb responses to acidic environmentsin cellulo. We report high‐content imaging modalities to probe Mtb sensing of external pH or visualise in real‐time Mtb intrabacterial pH within infected human macrophages. We discuss various methodologies with step‐by‐step analyses that enable robust image‐based quantifications. Finally, we highlight the advantages and limitations of these different approaches and discuss potential alternatives that can be applied to further investigate Mtb–host cell interactions. These methods can be adapted to study host–pathogen interactions in different biological systems and experimental settings. Altogether, these approaches represent a valuable tool to further broaden our understanding of the cellular and molecular mechanisms underlying intracellular pathogen survival.