Phase condensation of biomolecular particles is increasingly examined for its relevance in physiological contexts. When we observed formation of NEMO puncta associated with the outer mitochondrial membrane of damaged mitochondria, we wondered whether these puncta were incorporated into phase separated particles. Three standards of the field for determining phase separation are: (A) particles undergo fission and fusion like droplets of water, (B) individual puncta recover fluorescence after photobleaching, and (C) particles dissipate upon exposure to the organic compound 1,6-Hexanediol. Our system wherein NEMO puncta appeared to be attached to fragmented mitochondria precluded a systematic assessment of standard A. I.e., one NEMO punctum would so rarely encounter another, nor would it be prompted to split, that we could not confirm or rule out the potential for fission and fusion of droplets. This protocol describes our approach to investigating standards B and C.