Formaldehyde (FA) is a reactive carbon species that plays diverse biochemical roles in physiology and disease across all kingdoms of life. This one-carbon aldehyde is a ubiquitous environmental toxin, where its sources include vehicle exhaust and forest fires, and in the chemical industry in the formulation of household products like plywood and resins. Indeed, the Environmental Protection Agency (EPA) has designated FA as a human carcinogen with the Occupational Safety and Health Administration (OSHA) setting permissible exposure limits of 0.75 ppm in workplace air. Elevations in FA are associated with serious diseases, including cancer, our versatile 2-aza-Cope reactivity approach which enables selective detection of FA over other biologically relevant reactive carbon species, while other hydrazine-based mitochondrial aldehyde probes, that lack the rearrangement step, may interact with other aldehyde species. RFAPs provide a ratiometric readout (lex = 488 nm product/lex = 405 nm reactant) that enables internal self-calibration to correct for potential fluctuations in dye loading, light intensity, and sample thickness between biological specimens. Working through a modular synthetic pathway that relies on a key intermediate, RFAP-Alkyne (4), we synthesized and tested two MitoRFAP derivatives that leverage a triphenylphosphonium headgroup popularized by Murphy for mitochondrial targeting, finding that tuning to the appropriate linker length is key to optimizing the live-cell permeability of the probe. We establish the benefits of subcellular targeting by showing that MitoRFAP-2 can identify a depletion in mitochondrial FA pools in response to genetic knockout of SHMT enzymes, while an analogous, non-targeted RFAP-2 derivative is not capable of detecting this change to the mitochondrial 1C pool. We also leveraged MitoRFAP-2 to observe enhanced FA clearance achieved upon pharmacological activation of cells carrying the common human mutant form of ALDH2 (ALDH2*2) to correct for their genetic deficiencies in ALDH2 activity. To probe mitochondrial formaldehyde pools in living cells, we designed bifunctional fluorophores comprised of two components: (i) a formaldehyde-sensitive, coumarin dye that responds to FA through a 2-aza-Cope reaction, and (ii) a lipophilic triphenylphosphonium (TPP) cation as a mitochondrial-targeting moiety. We utilized a modular synthetic route that relied on a key intermediate, RFAP-alkyne (4), which was ligated to TPP-azide conjugates with varying linker lengths.