Macrophages are immune cells crucial in clearing our tissues from bacteria, viruses, dying cells, cell debris and other waste products. They also regulate inflammation by differentiating from non-activated (M0) cells that initiate inflammation (pro-inflammatory macrophages, M1), or resolve inflammation (anti-inflammatory macrophages, M2) macrophages. One of their key functions is to ingest pathogens within vesicles where they are degraded. The production of free radical (FR) plays an important role in this degradation process but also in macrophage differentiation and signaling. Here we used diamond-based quantum sensing to track free radical changes in vesicles with nanoscale resolution. We further followed the oxidative stress status, through free radical measurement during the macrophage activation process. We found that the three macrophage subtypes differed significantly in free radical generation in their vesicles. Additionally, we showed that the FR generation evolves over time in the different subtypes. We observed a 50% increase in radical production in M0 after 24 h compared to the T1 values measured after 4 h of cell culture, a decrease in M1 and constant radical levels in M2 macrophages. STATEMENT OF SIGNIFICANCE: Here we use quantum sensing for the first time to investigate the role that free radicals play in immune cells when they differentiate to fulfill their functions in the immune system. We were able to measure free radical generation specifically in vesicles while the macrophages differentiate.