The quaternary structure and dynamics of small heat shock proteins, such as Hsp27, underline their molecular chaperone function. We have previously established the structure-function relationship of phosphorylated Hsp27, whereby the dissociation of oligomers and chaperone activity are enhanced with this post-translational modification (1). As a result, it is proposed that the dimer is the chaperone active unit of Hsp27. However, it is unclear whether the dissociation of oligomer into dimers (i.e. changes in quaternary structure) or phosphorylation induced changes in the conformation and dynamics of the dimer itself (i.e. changes in tertiary structure), or both, are responsible for the enhanced chaperone activity of Hsp27. Therefore, the aims of this study were to explore the impact post-translational modifications, primarily phosphorylation, have on the tertiary and quaternary structure, and dynamics, of Hsp27. Using mutations that mimic phosphorylation, the structure and unfolding dynamics of Hsp27 dimers and higher-order oligomers were examined using ion mobility – mass spectrometry (IM-MS). Our results show that successive substitutions that mimic phosphorylation at serine residues do not significantly alter the quaternary structure of Hsp27 oligomers. Interestingly, IM-MS revealed that Hsp27 dimers are become progressively more unstructured and more resistant to unfolding in the gas-phase as the number of sites of phosphorylation increase. Together, our data suggests that oligomer dissociation and plasticity of the dimer both contribute to the enhanced chaperone activity of phosphorylated Hsp27. Post-translational modifications play a crucial role in modulating the tertiary and quaternary structure of Hsp27, and this is pivotal to its function as a protein stability sensor.