With data showing that MitoRFAP-2 can detect basal levels of mitochondrial FA and changes upon genetic and pharmacological alterations in mitochondrial FA sources and scavengers, we profiled basal mitochondrial FA levels across a panel of breast cancer cell lines with varying levels of expression of SHMT2 and ALDH2, two key regulators of formaldehyde metabolism in the mitochondria. We showcased the use of MitoRFAP-2 in this assay using flow cytometry, establishing that the probe can be used beyond microscopy applications. Cells were stained with MitoRFAP-2, suspended, and then analyzed. The results show that high levels of SHMT2 including closely related aldehydic analytes like acetaldehyde and methylglyoxal, and is sufficiently sensitive to not only demonstrate dose-dependent response to exogenous additions of FA, but to detect basal, endogenous levels of mitochondrial FA and respond to changes in this pool by genetic or pharmacological manipulation. For example, in an HCT-116 colorectal carcinoma cell line model, MitoRFAP-2 was able to decipher the contributions of serine hydroxymethyltransferase enzymes (SHMTs) as 1C sources feeding into the mitochondrial FA pool. Indeed, because upregulation of mitochondrially-localized SHMT2 is correlated with poor prognosis of cancer, with data showing that it drives cell proliferation in several cancer types, our findings connect this association with potentially aberrant elevations in mitochondrial FA. Importantly, the impact of genetic knockout of SHMT on mitochondrial FA pools that is identified by MitoRFAP-2, which cannot be detected by the untargeted parent probe RFAP-2, showcases the benefits of subcellular targeting of chemical imaging probes to increase their sensitivity and spatial resolution. Our data suggest that uniquely regulated, local fluxes of FA exist within subcellular compartments and presage that dynamic FA gradients may exist between various organelles within the cell.