Fused in sarcoma (FUS) is involved in the formation of nuclear biomolecular condensates associated with poly(ADP-ribose) [PAR] synthesis catalyzed by a DNA damage sensor such as PARP1. Here, we studied FUS microphases’ separation induced by poly(ADP-ribosyl)ated PARP1WT [PAR-PARP1WT] or its mutants PARP1Y986S and PARP1Y986H respectively synthesizing (short PAR)-PARP1Y986S or (short hyperbranched PAR)-PARP1Y986H using dynamic light scattering, fluorescence microscopy, turbidity assays, and atomic force microscopy. We observed that biologically relevant cations such as Mg2+, Ca2+, or Mn2+ or polyamines (spermine4+ or spermidine3+) were essential for the assembly of FUS with PAR-PARP1WT and FUS with PAR-PARP1Y986S in vitro. We estimated the range of the FUS-to-PAR-PARP1 molar ratio and the cation concentration that are favorable for the stability of the protein’s microphase-separated state. We also found that FUS microphase separation induced by PAR-PARP1Y986H (i.e., a PARP1 mutant attaching short hyperbranched PAR to itself) can occur in the absence of cations. The dependence of PAR-PARP1–induced FUS microphase separation on cations and on branching of PAR structure points to a potential role of the latter in the regulation of formation of FUS-related biological condensates and requires further investigation.