Huntington’s Disease, known for the presence of extended polyQ repeats in the huntingtin protein, has pathological effects on cellular membrane organelles. Here, we describe disturbances in membrane organization caused by the expression of mutant polyQ. We use the environment-sensitive fluorescent probe, LAURDAN, to assess variations in membrane lipid order of SH-SY5Y cells. The cells were induced to express labeled non-pathogenic (Q18mApple) and pathogenic (Q53mApple) proteins. Our approach takes advantage of LAURDAN’s affinity for hydrophobic regions, such as membranes, where it displays a red shift in emission associated with higher membrane fluidity (MF) instigated by changes in dipolar relaxation (DR) from the penetration of water molecules into the lipid bilayer. To assess fluidity in membranes, we use the Phasor analysis where we analyze LAURDAN fluorescence lifetime. In the phasor analysis plot, we identify two axes, one sensitive to MF and another to DR. Here we show that expression of pathogenic polyQ, correlates with increasing membrane fluidity, with no changes in DR processes, that suggests a disturbance in water penetration but not in membrane-lipid composition. Moreover, we show MF and DR processes are not inversely proportional and can be distinguished apart using lifetime measurements.