The phase-separating ability of Splicing Factor Proline- and Glutamine-rich (SPFQ), which leads to the formation of condensates, is attributed to its C-terminal Intrinsically Disordered Region (IDR). While this phase-separating domain has been identified, the specific sequence motifs within this domain that drive phase separation remain unknown. To investigate, a structure-guided approach using AlphaFold and other disorder prediction software such as IUPred, CatGranule, and PONDR, was employed to design various amino acid substitutions within the C-terminal IDR. Once these recombinant mutants were purified, the phase-separating ability of the mutants was determined via biophysical analysis, specifically microscopy. Some mutants, particularly those targeting arginine and tyrosine, drastically altered the phase-separating ability of SPFQ, with no droplets forming, even at concentrations nine times greater than the saturation concentration of wild-type SFPQ. In comparison, mutants targeting glycine residues altered the molecular dynamics of the formed droplets. Consistent with existing literature, different amino acids play distinct roles in driving phase separation. Specifically, amino acids that facilitate cation-pi interactions are more critical for facilitating phase separation, while others, like glycine, influence the physical properties of the droplets. Understanding the role of different amino acids in phase separation is crucial for predicting the impact of disease-related mutants on physiological phase separation and for designing synthetic phase-separating proteins for therapeutic applications.