Here we are presenting a comparative analysis of conformational changes of two amyloid β peptides, Aβ(25-35) and Aβ(1-42), in the presence and absence of a phospholipid system, namely, POPC/POPS (1-palmitoyl-2-oleoylphospatidylcholine/palmitoyl-2-oleoylphospatidylserine), through Raman spectroscopy, synchrotron radiation micro Fourier-transform infrared spectroscopy, and micro X-ray diffraction. Ringlike samples were obtained from the evaporation of pure and mixed solutions of the proteins together with the POPC/POPS system on highly hydrophilic substrates. The results confirm the presence of a α-helical to β-sheet transition from the internal rim of the ringlike samples to the external one in the pure Aβ(25-35) residual, probably due to the convective flow inside the droplets sitting on highly hydrophilic substrates enhancing the local concentration of the peptide at the external edge of the dried drop. In contrast, the presence of POPC/POPS lipids in the peptide does not result in α-helical structures and introduces the presence of antiparallel β-sheet material together with parallel β-sheet structures and possible β-turns. As a control, Aβ(1-42) peptide was also tested and shows β-sheet conformations independently from the presence of the lipid system. The μXRD analysis further confirmed these conclusions, showing how the absence of the phospholipid system induces in the Aβ(25-35) a probable composite α/β material while its coexistence with the peptide leads to a not oriented β-sheet conformation. These results open interesting scenarios on the study of conformational changes of Aβ peptides and could help, with further investigations, to better clarify the role of enzymes and alternative lipid systems involved in the amyloidosis process of Aβ fragments.