In vivo in time‐lapse microscopy experiments, DNA damage‐induced 53BP1 compartments showed droplet‐like behavior and underwent frequent fusion and occasional fission events (morphology, paricle size and count). Moreover, addition of the aliphatic alcohol 1,6‐hexanediol, resulted in disassembly of 53BP1 foci. After employing CRISPR/Cas9 to engineer the endogenous 53BP1 locus and integrate an in‐frame sequence encoding for the small monomeric red fluorescent protein mScarlet, the resulting fusion protein could be visualized by fluorescence microscopy. The fusion protein did not affect the cell cycle, localized to γH2AX‐positive sites of DNA damage (protein localization), showed the typical cell cycle‐regulated pattern of 53BP1 accumulation, and was sensitive to siRNA treatment targeted against 53BP1, confirming the role of 53BP1 in the formation of the observed compartments. Consistent with prior results, a short hyperosmotic challenge led to disassembly of 53BP1‐mScarlet compartments without affecting γH2AX accumulation. More importantly, however, live cell experiments with endogenously tagged 53BP1 expressed from its native promoter confirmed the dynamic, droplet‐like morphology of 53BP1 assemblies, their spherical shape, and their frequent fusions and fissions. 53BP1 nuclear bodies, occurring spontaneously and at enhanced frequency upon mild replication stress by low‐dose aphidicolin (APH) and ATR inhibitor (ATRi) treatment, showed droplet‐like behavior as well, and underwent frequent fusion events. In order to directly test whether 53BP1 possesses the capacity to phase separate, a system based on mCherry‐labeled Arabidopsis photoreceptor cryptochrome 2 (Cry2) fusion proteins was used to measure target protein optoDroplet formation in living cells. A Cry2‐53BP1 fusion resulted in rapid, light‐induced optoDroplet formation. Introducing a single amino acid mutation (W1495A) within the 53BP1 tandem tudor domain (TTD) to abrogate potentially confounding effects from TTD chromatin and protein interactions, and to assess the intrinsic capacity of 53BP1 to phase separate, further enhanced light‐induced optoDroplet formation. A series of deletion mutants used to identify the sequence elements driving 53BP1 phase separation revealed that the C‐terminus, comprising amino acids 1140–1972, was sufficient for optoDroplet formation, while the largely unstructured N‐terminus of 53BP1 was not, and that the oligomerization domain (OD) was critically involved. The 53BP1 C‐terminus is highly enriched for Arg and Tyr residues, implying that preferential optoDroplet formation of this region relies on cation-pi interactions (PMID:31267591). In vitro, the purified 53BP1 C‐terminus showed condensation into μm‐sized droplets in presence of Ficoll (morphology, particle size and count), and 53BP1 condensates co‐assembled double strand break‐mimicking fluorescently labeled DNA (PMID:31267591).