Membranous organelles are major endogenous sources of reactive oxygen and nitrogen species. When present at high levels, these species can cause macromolecular damage and disease. To better detect and scavenge free radical forms of the reactive species at their sources, we investigated whether nitrone spin traps could be selectively targeted to intracellular membranes using a bioorthogonal imaging approach. Electron paramagnetic resonance imaging demonstrated that the novel cyclic nitrone 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO) could be used to target the nitrone moiety to liposomes composed of phosphatidyl choline. To test localization with authentic membranes in living cells, fluorophores were introduced via strain-promoted alkyne-nitrone cycloaddition (SPANC). Two fluorophore-conjugated alkynes were investigated: hexynamide-fluoresceine (HYA-FL) and dibenzylcyclooctyne-PEG4-5/6-sulforhodamine B (DBCO-Rhod). Computational and mass spectrometry experiments confirmed the cycloadduct formation of DBCO-Rhod (but not HYA-FL) with diC12PO in cell-free solution. Confocal microscopy of bovine aortic endothelial cells treated sequentially with diC12PO and DBCO-Rhod demonstrated clear localization of fluorescence with intracellular membranes. These results indicate that targeting of nitrone spin traps to cellular membranes is feasible, and that a bioorthogonal approach can aid the interrogation of their intracellular compartmentalization properties.