Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterised by the degeneration of motor neurons in the motor cortex and spinal cord. Many ALS-associated RNA-binding proteins contain Intrinsically Disordered Regions (IDRs) that facilitate an essential biological process called Liquid-Liquid Phase Separation (LLPS). LLPS refers to the reversible and controlled formation of liquid-like biomolecular condensates via the self-organisation of biomolecules. One RNA-binding protein implicated in ALS is Splicing Factor Proline- and Glutamine-rich (SFPQ). Previous studies have demonstrated that the SFPQ C-terminal IDR is essential for driving LLPS, where SFPQ co-localises to long non-coding RNA NEAT Paraspeckle Assembly Transcript 1 (NEAT1) to form paraspeckles. Paraspeckles are molecular hubs that facilitate many essential cellular processes commonly affected in ALS. Although the domain responsible for driving SFPQ LLPS has been identified, the specific features within the SFPQ C-terminal IDR that drive LLPS remain unknown. A series of substitution mutations that target various amino acids were designed, and the phase-separating ability of the mutant constructs was tested in vivo using cellular paraspeckle assays and in vitro using droplet assays with recombinant proteins. Substitutions of Arg and Tyr amino acids drastically decreased the phase-separating ability of SFPQ. In comparison, amino acids such as Pro and Gly altered the molecular dynamics of the protein droplets. This work contributes to understanding how amino acids with the SFPQ C-IDR facilitate LLPS. This work is particularly significant as phase separation underpins many biological functions, and aberrant phase separation is implicated in many neurodegenerative diseases and cancers.