To validate our newly developed FIDA analysis, we measured the thermodynamic stability of the WT and F94W mutant of αSyn, and PI3-SH3 fibrils using other available techniques including DSF (differential scanning fluorimetry), static light scattering (SLS) intensity, Thioflavin T fluorescence, and ultracentrifugation (UCF) followed by quantification of the monomer in the supernatant using UVabsorbance (Figure , Table ). Depolymerization curves obtained by each method were fitted to the isodesmic model with shared m-value to reduce its correlation with ΔG, making the quantitative comparison of individual fits in terms of the latter parameter more reliable and straightforward. The isodesmic model is conceptually simpler than the cooperative model which limits overfitting by reducing the number of free parameters, particularly in cases where only a single protein concentration is measured . We obtained excellent agreement between experiments for the F94W αSyn with nearly overlapping depolymerization curves (apart from that obtained by ThT fluorescence) and differences between ΔG values within the range of fitting errors (Figure a, Table ). Since both FIDA and DSF are based on the detection of intrinsic fluorescence, we decided to test their limits by repeating the experiment using fibrils of wild type αSyn which does not contain tryptophan, but only 4 tyrosine residues as fluorophores (Figure , Table ). The fibrils were prepared according to the same protocol used for the F94W mutant and depolymerized at two-times higher final concentration (40 μM) to compensate for the signal loss. The sensitivity of the FIDA proved to be sufficient and we were able to obtain a well-defined depolymerization curve (Figure ). In contrast, DSF which utilizes the shift of the tryptophan fluorescence emission spectrum induced by changes in its local environment (e.g., monomeric vs fibrillar state ) did not yield any meaningful results (Figure ), confirming that the tyrosine residues lack the environmental sensitivity necessary to distinguish aggregated from soluble states. Interestingly, the stability of WT fibrils was ca. 5 kJ.mol -1 lower compared to the F94W mutant (Table ). This difference is higher than the variability of ΔG values obtained for WT using different techniques (~3 kJ.mol -1 ). Arguably, the mutation alters the energy landscape of αSyn and is therefore not a non-invasive probe for analysis of αSyn fibril stability. This is an important finding since similar effects might be, in principle, observed in studies of amyloid forming proteins lacking tryptophan and that use engineered Trp variants instead.