The TAR DNA-binding protein of 43 kDa (TDP-43) has been identified as the main component of amyotrophic lateral sclerosis (ALS) cytoplasmic inclusions. The link between this proteinopathy and TDP-43′s intrinsically disordered C-terminal domain is well known, but recently also, this domain has been shown to be involved in the formation of the membraneless organelles that mediate TDP-43′s functions. The mechanisms that underpin the liquid-liquid phase separation (LLPS) of these membraneless organelles undergo remain elusive. Crucially though, these factors may be the key to understanding the delicate balance between TDP-43′s physiological and pathological functions. In this study, we used nuclear magnetic resonance spectroscopy and optical methods to demonstrate that an α-helical component in the centre (residues 320–340) of the C-terminal domain is related to the protein's self-association and LLPS. Systematically analysing ALS-related TDP-43 mutants (G298S, M337V, and Q331K) in different buffer conditions at different temperatures, we prove that this phase separation is driven by hydrophobic interactions but is inhibited by electrostatic repulsion. Based on these findings, we rationally introduced a mutant, W334G, and demonstrate that this mutant disrupts LLPS without disturbing this α-helical propensity. This tryptophan may serve as a key residue in this protein's LLPS.