Fluorescent nanodiamonds have exceptional optical properties and are highly biocompatible, which allows to use them as labels for long-term tracking of the cells. The research fields that make use of this application of nanodiamonds include stem cell biology and cancer biology, where quiescent and differentiating cells can be traced in vitro and in vivo. However, these studies focus on using nanodiamonds as simple labels, whereas they can serve as highly sensitive intracellular sensors for free radical species. In this chapter, we aimed to bring the two approaches together and to assess the free radical production in the cells over the course of their differentiation. We report on the successful enterocytic differentiation of HT-29 colon adenocarcinoma cells, pre-loaded with fluorescent nanodiamonds. The cells were cultured in butyrate-free or butyrate-supplemented medium for 13 days. Butyrate-treated cells developed the morphological and molecular traits, characteristic for the normal enterocytes. Fluorescent nanodiamonds did not have a negative effect on the process of differentiation. Moreover, the particles could be found in the cytoplasm of both undifferentiated and re-differentiated cells even after 13 days of culture. The internalized nanodiamonds were used to assess the free radical load in the undifferentiated and re-differentiated HT-29 cells at different stages of the experiment. Consistently with previous findings, re-differentiated HT-29 cells showed higher free radical load than undifferentiated ones.