Lactate, the product of aerobic glycolysis, plays a dual role as fuel and intercellular signal in inflammation, immune evasion, and tumor progression. The production of lactate by macrophages has been associated with their polarization and function. Here we describe imaging protocols to characterize the metabolism of cultured human macrophages using a genetically encoded fluorescent sensor-specific for lactate. By superfusing cultures with increasing lactate concentrations and pharmacological inhibitors, it is possible to estimate the kinetic parameters of monocarboxylate transporter 4 (MCT4) and lactate production. Practical advice is given regarding sensor expression, imaging, and data analysis. The spatiotemporal resolution of this technique is amenable to the study of fast events at the single-cell level in different immune and other cell types.