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Amyloidogenic peptides aggregate through a well-defined pathway to form amyloid fibrils. Many amyloidogenic peptides are known to toxic through the formation of fibrillar, and pre-fibrillar aggregates. Amyloid-Beta is the most well researched amyloidogenic peptide due to its implications in the neurodegenerative disease, Alzheimer’s Disease. While the specific mechanisms for Amyloid Beta induced toxicity are not fully elucidated, membrane interactions are considered to be a significant component to their toxicity. Functional amyloids are amyloidogenic peptides that exhibit functional roles for the organism that produces it. Many peptide hormones are classed as functional amyloids as they form fibrils in the dense-core secretory granules. As more and more peptides are classified as functional amyloids, a gap in our understanding of what constitutes an amyloidogenic peptide to induce toxicity, or to elicit a non-toxic functional role became apparent. One theory proposes that the critical difference between the two amyloids are in the way they interact with membranes. Characterising membrane interactions in a live cell is exceptionally challenging to undertake due to the complex and dynamic nature of cellular membranes. Spectral phasor analysis can elucidate the spectral properties of individual fluorescent probes and can be used to correlate spectral changes with changes to the probes’ local microenvironment. Here spectral phasor analysis was tested to see if it can deduce changes in the spectral properties of the lipophilic dye Nile Red in the presence of monomeric and aggregated forms of Amyloid Beta and Neurokinin B. Spectral phasor analysis was done on Nile Red to see if the technique can elucidate discrete spectral properties based on its subcellular location. Discrete microenvironments expressed via λmax and spectral width of Nile Red emission spectra were isolated in the plasma membrane, perinuclear, and nuclear region. Using varying sized cursors confirm that minor spectral shifts in Nile Red emission were detectable via spectral phasor analysis. Analysis of plasma membrane Nile Red in the presence of monomeric Amyloid Beta and Neurokinin B reveal spectral shifts that became discrete over time. λmax values in the presence of both peptides were similar at 14 hrs but became distinct at 24 hrs. Shifts in emission spectra in the presence of aggregated Amyloid Beta and Neurokinin B showed an opposite trend with λmax values became similar over time. This confirms that the plasma membrane interacts differently to each peptide and its aggregate over time. Spectral phasor analysis can characterise spectral shifts of a fluorescent probe at a pixel by pixel resolution. Future development would allow for these discrete changes to be linked to a specific membrane interaction, allowing the use of spectral phasor analysis tied with Nile Red to determine the membrane interactions of other toxic and functional amyloidogenic peptides.